Massively parallel single cell analysis

ABSTRACT

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/472,363, filed on Aug. 28, 2014, which claims the benefit ofU.S. Provisional Application No. 62/012,237, filed on Jun. 13, 2014,U.S. Provisional Application No. 61/952,036, filed on Mar. 12, 2014, andU.S. Provisional Application No. 61/871,232, filed on Aug. 28, 2013. Allof the aforementioned priority applications are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 27, 2016, isnamed Sequence_Listing.txt and is 211,530 bytes in size

BACKGROUND

Multicellular masses, such as tissues and tumors, may comprise aheterogeneous cellular milieu. These complex cellular environments mayoften display multiple phenoytpes, which may be indicative of multiplegenotypes. Distilling multicellular complexity down to single cellvariability is an important facet of understanding multicellularheterogeneity. This understanding may be important in the development oftherapeutic regimens to combat diseases with multiple resistancegenotypes.

SUMMARY OF THE INVENTION

One aspect provided is a method, comprising obtaining a samplecomprising a plurality of cells; labeling at least a portion of two ormore polynucleotide molecules, complements thereof, or reaction productstherefrom, from a first cell of the plurality and a second cell of theplurality with a first same cell label specific to the first cell and asecond same cell label specific to the second cell; and a molecularlabel specific to each of the two or more polynucleotide molecules,complements thereof, or reaction products therefrom, wherein eachmolecular label of the two or more polynucleotide molecules, complementsthereof, or reaction products therefrom, from the first cell are uniquewith respect to each other, and wherein each molecular label of the twoor more polynucleotide molecules, complements thereof, or reactionproducts therefrom, from the second cell are unique with respect to eachother. In some embodiments, the method further comprises sequencing theat least a portion of two or more polynucleotide molecules, complementsthereof, or reaction products therefrom. In some embodiments, the methodfurther comprises analyzing sequence data from the sequencing toidentify a number of individual molecules of the polynucleotides in aspecific one of the cells. In some embodiments, the cells are cancercells. In some embodiments, the cells are infected with viralpolynucleotides. In some embodiments, the cells are bacteria or fungi.In some embodiments, the sequencing comprises sequencing with readlengths of at least 100 bases. In some embodiments, the sequencingcomprises sequencing with read lengths of at least 500 bases. In someembodiments, the polynucleotide molecules are mRNAs or micro RNAs, andthe complements thereof and reaction products thereof are complements ofand reaction products therefrom the mRNAs or micro RNAs. In someembodiments, the molecular labels are on a bead. In some embodiments,the label specific to an individual cell is on a bead. In someembodiments, the label specific to an individual cell and the molecularlabels are on beads. In some embodiments, the method is performed atleast in part in an emulsion. In some embodiments, the method isperformed at least in part in a well or microwell of an array. In someembodiments, the presence of a polynucleotide that is associated with adisease or condition is detected. In some embodiments, the disease orcondition is a cancer. In some embodiments, at least a portion of amicroRNA, complement thereof, or reaction product therefrom is detected.In some embodiments, the disease or condition is a viral infection. Insome embodiments, the viral infection is from an enveloped virus. Insome embodiments, the viral infection is from a non-enveloped virus. Insome embodiments, the virus contains viral DNA that is double stranded.In some embodiments, the virus contains viral DNA that is singlestranded. In some embodiments, the virus is selected from the groupconsisting of a pox virus, a herpes virus, a vericella zoster virus, acytomegalovirus, an Epstein-Barr virus, a hepadnavirus, a papovavirus,polyomavirus, and any combination thereof. In some embodiments, thefirst cell is from a person not having a disease or condition and thesecond cell is from a person having the disease or condition. In someembodiments, the persons are different. In some embodiments, the personsare the same but cells are taken at different time points. In someembodiments, the first cell is from a person having the disease orcondition and the second cell is from the same person. In someembodiments, the cells in the sample comprise cells from a tissue ororgan. In some embodiments, the cells in the sample comprise cells froma thymus, white blood cells, red blood cells, liver cells, spleen cells,lung cells, heart cells, brain cells, skin cells, pancreas cells,stomach cells, cells from the oral cavity, cells from the nasal cavity,colon cells, small intestine cells, kidney cells, cells from a gland,brain cells, neural cells, glial cells, eye cells, reproductive organcells, bladder cells, gamete cells, human cells, fetal cells, amnioticcells, or any combination thereof.

One aspect provided is a solid support comprising a plurality ofoligonucleotides each comprising a cellular label and a molecular label,wherein each cellular label of the plurality of oligonucleotides are thesame, and each molecular label of the plurality of oligonucleotides aredifferent; and wherein the solid support is a bead, the cellular labelis specific to the solid support, the solid support, when placed at thecenter of a three dimensional Cartesian coordinate system, hasoligonucleotides extending into at least seven of eight octants, or anycombination thereof. In some embodiments, the plurality ofoligonucleotides further comprises at least one of a sample label; auniversal label; and a target nucleic acid binding region. In someembodiments, the solid support comprises the target nucleic acid bindingregion, wherein the target nucleic acid binding region comprises asequence selected from the group consisting of a gene-specific sequence,an oligo-dT sequence, a random multimer, and any combination thereof. Insome embodiments, the solid support further comprises a target nucleicacid or complement thereof. In some embodiments, the solid supportcomprises a plurality of target nucleic acids or complements thereofcomprising from about 0.01% to about 100% of transcripts of atranscriptome of an organism or complements thereof, or from about 0.01%to about 100% of genes of a genome of an organism or complementsthereof. In some embodiments, the cellular labels of the plurality ofoligonucleotides comprise a first random sequence connected to a secondrandom sequence by a first label linking sequence; and the molecularlabels of the plurality of oligonucleotides comprise random sequences.In some embodiments, the solid support is selected from the groupconsisting of a polydimethylsiloxane (PDMS) solid support, a polystyrenesolid support, a glass solid support, a polypropylene solid support, anagarose solid support, a gelatin solid support, a magnetic solidsupport, a pluronic solid support, and any combination thereof. In someembodiments, the plurality of oligonucleotides comprise a linkercomprising a linker functional group, and the solid support comprises asolid support functional group; wherein the solid support functionalgroup and linker functional group connect to each other. In someembodiments, the linker functional group and the solid supportfunctional group are individually selected from the group consisting ofC6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), andany combination thereof. In some embodiments, molecular labels of theplurality of oligonucleotides comprise at least 15 nucleotides.

One aspect provided is a kit comprising any of the solid supportsdescribed herein, and instructions for use. In some embodiments, the kitfurther comprises a well. In some embodiments, the well is comprised inan array. In some embodiments, the well is a microwell. In someembodiments, the kit further comprises a buffer. In some embodiments,the kit is contained in a package. In some embodiments, the package is abox. In some embodiments, the package or box has a volume of 2 cubicfeet or less. In some embodiments, the package or box has a volume of 1cubic foot or less.

One aspect provided is an emulsion comprising any of the solid supportsdescribed herein.

One aspect provided is a composition comprising a well and any of thesolid supports described herein.

One aspect provided is a composition comprising a cell and any of thesolid supports described herein.

In some embodiments, the emulsion or composition further comprises acell. In some embodiments, the cell is a single cell. In someembodiments, the well is a microwell. In some embodiments, the microwellhas a volume ranging from about 1,000 μm³ to about 120,000 μm³.

One aspect provided is a method, comprising contacting a sample with anysolid support disclosed herein, hybridizing a target nucleic acid fromthe sample to an oligonucleotide of the plurality of oligonucleotides.In some embodiments, the method further comprises amplifying the targetnucleic acid or complement thereof. In some embodiments, the methodfurther comprises sequencing the target nucleic acid or complementthereof, wherein the sequencing comprises sequencing the molecular labelof the oligonucleotide to which the target nucleic acid or complementthereof is bound. In some embodiments, the method further comprisesdetermining an amount of the target nucleic acid or complement thereof,wherein the determining comprises quantifying levels of the targetnucleic acid or complement thereof; counting a number of sequencescomprising the same molecular label; or a combination thereof. In someembodiments, the method does not comprise aligning any same molecularlabels or any same cellular labels. In some embodiments, the amplifyingcomprises reverse transcribing the target nucleic acid. In someembodiments, the amplifying employs a method selected from the groupconsisting of: PCR, nested PCR, quantitative PCR, real time PCR, digitalPCR, and any combination thereof. In some embodiments, the amplifying isperformed directly on the solid support; on a template transcribed fromthe solid support; or a combination thereof. In some embodiments, thesample comprises a cell. In some embodiments, the cell is a single cell.In some embodiments, the contacting occurs in a well. In someembodiments, the well is a microwell and is contained in an array ofmicrowells.

One aspect provided is a device, comprising a plurality of microwells,wherein each microwell of the plurality of microwells has a volumeranging from about 1,000 μm³ to about 120,000 μm³. In some embodiments,each microwell of the plurality of microwells has a volume of about20,000 μm³. In some embodiments, the plurality of microwells comprisesfrom about 96 to about 200,000 microwells. In some embodiments, themicrowells are comprised in a layer of a material. In some embodiments,at least about 10% of the microwells further comprise a cell. In someembodiments, the device further comprises any of the solid supportsdescribed herein.

One aspect provided is an apparatus comprising any of the devicesdescribed herein, and a liquid handler. In some embodiments, the liquidhandler delivers liquid to the plurality of microwells in about onesecond. In some embodiments, the liquid handler delivers liquid to theplurality of microwells from a single input port. In some embodiments,the apparatus further comprises a magnet. In some embodiments, theapparatus further comprises at least one of: an inlet port, an outletport, a pump, a valve, a vent, a reservoir, a sample collection chamber,a temperature control apparatus, or any combination thereof. In someembodiments, the apparatus comprises the sample collection chamber,wherein the sample collection chamber is removable from the apparatus.In some embodiments, the apparatus further comprises an optical imager.In some embodiments, the optical imager produces an output signal whichis used to control the liquid handler. In some embodiments, theapparatus further comprises a thermal cycling mechanism configured toperform a polymerase chain reaction (PCR) amplification ofoligonucleotides.

One aspect provided is a method of producing a clinical diagnostic testresult, comprising producing the clinical diagnostic test result withany device or apparatus described herein; any solid support describedherein; any method described herein; or any combination thereof. In someembodiments, the clinical diagnostic test result is transmitted via acommunication medium.

One aspect provided is a method of making any of the solid supportsdescribed herein, comprising attaching to a solid support: a firstpolynucleotide comprising a first portion of the cellular label, and afirst linker; andcontacting a second polynucleotide comprising a secondportion of the cellular label, a sequence complementary to the firstliker, and the molecular label. In some embodiments, the thirdpolynucleotide further comprises a target nucleic acid binding region.

In some embodiments, an emulsion, microwell, or well contains only onecell. In some embodiments, from 1 to 2,000,000 emulsions, microwells, orwells each contain only one cell. In some embodiments, the methodcomprises distributing at most one cell into each emulsion, microwell,or well. In some embodiments, a single solid support and a single cellare distributed to an emulsion, microwell, or well. In some embodiments,from 1 to 2,000,000 emulsions, microwells, or wells each havedistributed thereto one cell and one solid support. In some embodiments,the method comprises distributing at most one solid support peremulsion, microwell, or well. In some embodiments, the method comprisesdistributing one solid support and one cell to each of from 1 to2,000,000 microwells, emulsions, or wells. In some embodiments, celldistribution is random or non-random. In some embodiments, celldistribution is stochastic. In some embodiments, a cell is distributedby a cell sorter. In some embodiments, a cell is distributed bycontacting one or more wells, microwells, or emulsions with a dilutesolution of cells diluted so that at most one cell is distributed to theone or more wells, microwells, or emulsions.

In some embodiments, the target specific regions, target specificregions of the plurality of oligonucleotides, or the target specificregion of the two or more polynucleotide molecules, comprise sequencescomplementary to two or more targets of a target panel. In someembodiments, the two or more targets of the target panel are biomarkers.In some embodiments, the biomarkers are biomarkers for a disease orcondition. In some embodiments, the disease or condition is a cancer, aninfection, a viral infection, an inflammatory disease, aneurodegenerative disease, a fungal disease, a bacterial infection, orany combination thereof. In some embodiments, the panel comprises from:2-50,000, 2-40,000, 2-30,000, 2-20,000, 2-10,000, 2-9000, 2-8,000,2-7,000, 2-6,000, 2-5,000, 2-1,000, 2-800, 2-700, 2-600, 2-500, 2-400,2-300, 2-200, 2-100, 2-75, 2-50, 2-40, 2-30, 2-20, 2-10, or 2-5biomarkers.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts an exemplary solid support conjugated with an exemplaryoligonucleotide. FIG. 1 discloses “dT(17)V” as SEQ ID NO: 829.

FIG. 2A-C depicts an exemplary workflow for synthesizing oligonucleotidecoupled beads using split-pool synthesis.

FIG. 3 depicts an exemplary oligonucleotide coupled bead. FIG. 3discloses “dT(17)V” as SEQ ID NO: 829.

FIG. 4 illustrates an exemplary embodiment of a microwell array.

FIG. 5 depicts an exemplary distribution of solid supports in amicrowell array.

FIG. 6A-C show exemplary distribution cells onto microwell arrays. FIG.6A shows the distribution of K562 cells (large cell size). FIG. 6B showsthe distribution of Ramos cells (small cell size). FIG. 6C shows thedistribution of Ramos cells and oligonucleotide coupled beads ontomicrowell arrays, with solid arrows pointing to the Ramos cells anddashed arrows pointing to the oligonucleotide coupled beads.

FIG. 7 shows exemplary statistics of the microwell volume, solid supportvolume, and amount of biological material obtained from lysis.

FIG. 8A-C illustrates an exemplary embodiment of bead cap sealing. FIG.8A-B show images of a microarray well with cells and oligonucleotidebeads distributed into wells of a microarray well and with largersephadex beads used to seal the wells. Dotted arrows point to the cells,dashed arrows point to the oligonucleotide coupled beads and the solidarrows point to the sephadex beads. FIG. 8C depicts a schematic of thecell and oligonucleotide bead (e.g., oligobead) deposited within a wellwith a sephadex bead used to seal the well.

FIG. 9 depicts a bar graph comparing amplification efficiency of GAPDHand RPL19 amplified from microwells and tubes. The grey bars representdata from the microwell. The white bars represent data from the tube.

FIG. 10 depicts an agarose gel comparing amplification specificity ofthree different genes directly on a solid support.

FIG. 11A-I show graphical representations of the sequencing results.

FIG. 12A-C show a histogram of the sequencing results for the K562-onlysample, Ramos-only sample, and K562+Ramos mixture sample, respectively.

FIG. 12D-E shows a graph of the copy number for genes listed in Table 3for the Ramos-only cell sample and K562-only cell sample, respectively.

FIG. 12F-I show the copy number for individual genes.

FIG. 12J-M show graphs of the number of unique molecules per gene(y-axis) for the beads with the 100 unique barcode combinations.

FIG. 12N-O show enlarged graphs of two beads that depict the generalpattern of gene expression profiles for the two cell types.

FIG. 12P shows a scatter plot of results based on principal componentanalysis of gene expression profile of 768 beads with >30 molecules perbead from the K562+Ramos mixture sample.

FIG. 12Q-R show histograms of the copy number per amplicon per bead forthe K562-like cells (beads on the left of the first principal componentbased on FIG. 12P) and Ramos-like cells (beads on the right of the firstprincipal component based on FIG. 12P), respectively.

FIG. 12S-T show the copy number per bead or single cell of theindividual genes for the K562-like cells (beads on the left of the firstprincipal component based on FIG. 12P) and Ramos-like cells (beads onthe right of the first principal component based on FIG. 12P),respectively.

FIG. 13A depicts general gene expression patterns for the mouse andRamos cells.

FIG. 13B-C show scatter plots of results based on principal componentanalysis of gene expression profile of the high density sample and lowdensity sample, respectively.

FIG. 13D-E depict graphs of the read per barcode (bc) combination(y-axis) versus the unique barcode combination, sorted by the totalnumber of molecules per bc combination (x-axis) for Ramos-like cells andmouse-like cells from the high density sample, respectively.

FIG. 13F-G depict graphs of the number of molecules per barcode (bc)combination (y-axis) versus the unique barcode combination, sorted bythe total number of molecules per bc combination (x-axis) for Ramos-likecells and mouse-like cells from the high density sample, respectively.

FIG. 13H-I depict graphs of the read per barcode (bc) combination(y-axis) versus the unique barcode combination, sorted by the totalnumber of molecules per barcode combination (x-axis) for Ramos-likecells and mouse-like cells from the low density sample, respectively.

FIG. 13J-K depict graphs of the number of molecules per barcodecombination (y-axis) versus the unique barcode combination, sorted bythe total number of molecules per barcode combination (x-axis) forRamos-like cells and mouse-like cells from the low density sample,respectively.

FIG. 14 shows a graph depicting the genes on the X-axis and the log 10of the number of reads.

FIG. 15A shows a graph of the distribution of genes detected perthree-part cell label (e.g., cell barcode). FIG. 15B shows a graph ofthe distribution of unique molecules detected per bead (expressing thegene panel).

FIG. 16 depicts the cell clusters based on the genes associated with acell barcode.

FIG. 17A-D show the analysis of monocyte specific markers. FIG. 17Eshows the cell cluster depicted in FIG. 16.

FIG. 18A-B show the analysis of the T cell specific markers. FIG. 18Cshows the cell cluster depicted in FIG. 16.

FIG. 19A-B show the analysis of the CD8+ T cell specific markers. FIG.19C shows the cell cluster depicted in FIG. 16.

FIG. 20A shows the analysis of CD4+ T cell specific markers. FIG. 20Bshows the cell cluster depicted in FIG. 16.

FIG. 21A-D show the analysis of Natural Killer (NK) cell specificmarkers. FIG. 21E shows the cell cluster depicted in FIG. 16.

FIG. 22A-E show the analysis of B cell specific markers. FIG. 22F showsthe cell cluster depicted in FIG. 16.

FIG. 23A-F show the analysis of Toll-like receptors. Toll-like receptorsare mainly expressed by monocytes and some B cells. FIG. 23G shows thecell cluster depicted in FIG. 16.

FIG. 24 depicts a graph of the genes versus the log 10 of the number ofreads.

FIG. 25A-D shows graphs of the molecular barcode versus the number ofreads or log 10 of the number of reads for two genes.

FIG. 26A shows a graph of the number of genes in the panel expressed percell barcode versus the number of unique cell barcodes/single cell. FIG.26B shows a histogram of the number of unique molecules detected perbead versus frequency of the number of cells per unique cell barcodecarrying a given number of molecules. FIG. 26C shows a histogram of thenumber of unique GAPDH molecules detected per bead versus frequency ofthe number of cells/unique cell barcode carrying a given number ofmolecules.

FIG. 27 shows a scatterplot of the 856 cells.

FIG. 28 shows a heat map of expression of the top 100 (in terms of thetotal number of molecules detected).

FIG. 29 shows a workflow for Example 12.

FIG. 30 shows a workflow for Example 13. FIG. 30 discloses “dT(17)V” asSEQ ID NO: 829 and “AAAAAAAAAA” as SEQ ID NO: 830.

FIG. 31A-C. Clustering of single cells in controlled mixtures containingtwo distinct cell types. FIG. 31A. Clustering of a 1:1 mixture of K562and Ramos cells by principal component analysis of the expression of 12genes. The biplot shows two distinct clusters, with one clusterexpressing Ramos specific genes and the other expressing K562 specificgenes. FIG. 31B. Principal component analysis of a mixture containing asmall percentage of Ramos cells in a background of primary B cells froma healthy individual using a panel of 111 genes. The color of each datapoint indicates the total number of unique transcript molecules detectedacross the entire gene panel. A set of 18 cells (circled) out of 1198cells displays a distinct gene expression profile and with much highertranscription levels. FIG. 31C. Heatmap showing expression level of eachgene in the top 100 cells in the sample of FIG. 31B, ranked by the totalnumber of transcript molecules detected in the gene panel. Genes areordered via hierarchical clustering in terms of correlation. The top 18cells, indicated by the horizontal red bar, expressed preferentially aset of genes known to be associated with follicular lymphoma, asindicated by the vertical red bar.

FIG. 31D. PCA analysis of primary B cells with spiked in Ramos cells.Color of each data point (single cell) indicates the log of the numberof transcript molecules each cell carries for the particular gene. Top 7rows: Genes that are preferentially expressed by the subset of 18 cellsthat are likely Ramos cells. First row genes (from left to right)include GAPDH, TCL1A, MKI67 and BCL6. Second row genes (from left toright) include MYC, CCND3, CD81 and GNAI2. Third row of genes (from leftto right) include IGBP1, CD20, BLNK and DOCKS. Fourth row of genes (fromleft to right) include IRF4, CD22, IGHM and AURKB. Fifth row of genes(from left to right) include CD38, CD10, LEFT and AICDA. Sixth row ofgenes (from left to right) include CD40, CD27, IL4R and PRKCD. Seventhrow of genes (from left to right) include RGS1, MCL1, CD79a and HLA-DRA.Last row: Genes that are expressed preferentially by a subset of primaryB cells but not especially enriched in those 18 cells. Genes in the lastrow (from left to right) include IL6, CD23a, CCR7 and CXCR5.

FIG. 32 Expression of GAPDH. Color indicates natural log of the numberof unique transcript molecules observed per cell.

FIG. 33A-F shows the principal component analysis (PCA) for monocyteassociated genes. FIG. 33A shows the PCA for CD16. FIG. 33B shows thePCA for CCRvarA. FIG. 33C shows the PCA for CD14. FIG. 33D shows the PCAfor S100A12. FIG. 33E shows the PCA for CD209. FIG. 33F shows the PCAfor IFNGR1.

FIG. 34A-B shows the principal component analysis (PCA) for pan-T cellmarkers (CD3). FIG. 34A shows the PCA for CD3D and FIG. 34B shows thePCA for CD3E.

FIG. 35A-E shows the principal component analysis (PCA) for CD8 T cellassociated genes. FIG. 35A shows the PCA for CD8A. FIG. 35B shows thePCA for EOMES. FIG. 35C shows the PCA for CD8B. FIG. 35D shows the PCAfor PRF1. FIG. 35E shows the PCA for RUNX3.

FIG. 36A-C shows the principal component analysis (PCA) for CD4 T cellassociated genes. FIG. 36A shows the PCA for CD4. FIG. 36B shows the PCAfor CCR7. FIG. 36C shows the PCA for CD62L.

FIG. 37A-F shows the principal component analysis (PCA) for B cellassociated genes. FIG. 37A shows the PCA for CD20. FIG. 37B shows thePCA for IGHD. FIG. 37C shows the PCA for PAX5. FIG. 37D shows the PCAfor TCL1A. FIG. 37E shows the PCA for IGHM. FIG. 37F shows the PCA forCD24.

FIG. 38A-C shows the principal component analysis (PCA) for NaturalKiller cell associated genes. FIG. 38A shows the PCA for KIR2DS5. FIG.38B shows the PCA for CD16. FIG. 38C shows the PCA for CD62L.

FIG. 39 Simultaneous identification of major cell types in a human PBMCsample (632 cells) by PCA analysis of 81 genes assayed by CytoSeq Cellswith highly correlated expression profile are coded with similar color.

FIG. 40A-B Correlation analysis of single cell gene expression profileof PBMC sample. 40A. A matrix showing the pairwise correlationcoefficient across 632 cells in the sample. The cells are ordered suchthat those with highly correlated gene expression profile are groupedtogether. FIG. 40B. Heatmap showing the expression of each gene by eachcell. The cells (columns) are ordered in the same manner as thecorrelation matrix above. The genes (rows) are ordered such that genesthat share highly similar expression pattern across the cells aregrouped together. The cell type of each cluster of cells may beidentified by the group of genes the cells co-expressed. Within eachmajor cell cluster, there is substantial degree of heterogeneity interms of gene expression.

FIG. 41 data represents that of 731 cells from a replicate experiment ofPBMC sample from the same donor. Cells with similar gene expressionprofile (based on hierarchical clustering using correlation coefficient)are plotted with similar color.

FIG. 42 shows a heat map demonstrating the correlation in geneexpression profile between genes.

FIG. 43 Description of CytoSeq. FIG. 43A. Experimental procedure forCytoSeq. FIG. 43B. Structure of oligonucleotides attached to beads.

FIGS. 44A-C illustrate dissecting sub-populations of CD3+ T cells. FIG.44A. PCA of Donor 1 unstimulated sample reveals two major branches ofcells. The expression level (log of unique transcript molecule) of aparticular gene within each cell is indicated with color. Helper T cellassociated cytokine and effector genes are enriched in cells in thelower branch, while cytotoxic T cell associated genes are enriched inthe upper branch. Shown here are representative genes. First row showshelper T cell related genes and include (from left to right) CD4, SELLand CCR7. Second row shows cytotoxic T cell related genes and include(from left to right) CD8A, NKG2D and EOMES. FIG. 44B. PCA of Donor 1anti-CD3/anti-CD28 stimulated sample showing enrichment of expression ofindicated genes to one of the two main branches representing helper andcytotoxic T cells. These genes are present at low amounts in theunstimulated sample. First two rows show genes that are known to beassociated with activated T cells and include (from left to right) inthe first row IRF4, CD69 and MYC and in the second row GAPDH, TNF andIFNG. The third row shows genes that are known to be associated withactivated helper T cells and include (from left to right) IL2, LTA andCD40LG. The fourth row shows genes that are known to be associated withactivated cytotoxic T cells and include (from left to right) CCL4, CCL3and GZMB. FIG. 44C. Number of cells that contribute to the overallexpression level of genes that exhibit large fold-changes when comparingstimulated over unstimulated samples in aggregate data. For severalcytokines (red arrows), the contribution from only a small number ofcells is responsible for large overall gene expression change in theentire population.

FIGS. 45A-C illustrate PCA plots of T cell samples that have undergonestimulation with anti-CD28/anti-CD3 beads in the two donors, and thecorresponding unstimulated samples, with emphasis on the expression ofgenes that clearly show preferential expression in either helper orcytotoxic subsets in the unstimulated samples. The color of each datapoint (single cell) indicates log(number of unique transcript molecule)per cell for the indicated gene. For each pair of stimulated andunstimulated graphs in each donor, the color range is adjusted to be thesame. FIG. 45A. Genes that are known to be associated with both helperand cytotoxic T cells. FIG. 45B. Genes that are known to be associatedwith cytotoxic T cells. FIG. 45C. Genes that are known to be associatedwith helper T cells.

FIG. 46A-D PCA plots of T cell samples that have undergone stimulationwith anti-CD28/anti-CD3 beads in the two donors, and the correspondingunstimulated samples, with emphasis on the expression of genes that areexpressed in the stimulated samples but at low or undetectable level inthe unstimulated samples. The color of each data point (single cell)indicates log(number of unique transcript molecule) per cell for theindicated gene. For each pair of stimulated and unstimulated graphs ineach donor, the color range is adjusted to be the same. 46A and 46D.Genes that are expressed by both branches of cells upon activation. 46B.Genes that are expressed preferentially by cells in the upper branchupon activation. These genes are known to be associated with activatedcytotoxic T cells. 46C. Genes that are expressed preferentially by cellsin the lower branch upon activation. These genes are known to beassociated with activated helper T cells.

FIG. 47 Clustering of data from Donor 1's unstimulated CD3+ T cellsshows separations of CD4 and CD8 cells, as well as a group of cells thatexpress Granzyme K and Granzyme A but little CD8. Top: Heatmap showingcorrelation between each pair of cells. Cells that are highly correlatedare grouped together. Bottom: Heatmap showing the level of expression ofeach gene of each cell. Cells and genes are ordered via bidirectionalhierarchical clustering.

FIG. 48. Similar to FIG. 47, but showing data from anti-CD3/anti-CD28stimulated CD3+ T cell sample of Donor 1. Top: Heatmap showingcorrelation between each pair of cells. Cells that are highly correlatedare grouped together. Bottom: Heatmap showing the level of expression ofeach gene of each cell. Cells and genes are ordered via bidirectionalhierarchical clustering.

FIG. 49A-C In donor 1, large overall fold change was observed forvarious cytokines in the antiCD28/antiCD3 stimulated sample, as comparedto the unstimulated one. FIGS. A-B: The large fold changes of thesecytokines were mostly contributed by only a few single cells (dots thatare enclosed with squares or circles). A number of these cytokines werecontributed by the same small number of cells. FIG. 49C: Theco-expression patterns of these cytokines coincide with the signaturecytokine combination for the Th2 and Th17 subsets of helper T cells.

FIG. 50A-B. Dissecting sub-populations of CD8+ T cells. FIG. 50A.Clustering of CytoSeq data defines two major groups of CD8+ cells—onegroup expresses genes shared by central memory/naive cells, and theother group expresses genes shared by effector memory/effector cells.Shown here is data of Donor 2's unstimulated sample. Top: Heatmapshowing correlation between each pair of cells. Bottom: Heatmap showingthe level of expression of each gene in each cell. Cells and genes areordered via bidirectional hierarchical clustering. FIG. 50B.Identification of rare antigen specific T cell by expression of gammainterferon (IFNG) in CD8+ T cells from two donors after stimulation withCMV peptide pool. Each cell is plotted on the 2D principal componentspace. Cells expressing IFNG (circled) are usually among those with themost total detected transcripts in the panel (indicated by the color).In donor 2, the top expressing cell (square) does not produce IFNG butexpresses cytokines IL6 and IL1B. Number next to each circle indicatesthe rank in descending order the number of total unique transcriptmolecules detected for that cell.

FIG. 51. Similar to FIG. 50A except the data here represents that ofDonor 2 CMV stimulated sample. A. Clustering of CytoSeq data defines twomajor groups of CD8+ cells—one group expresses genes shared by centralmemory/naive cells, and the other group expresses genes shared byeffector memory/effector cells. Shown here is data of Donor 2'sunstimulated sample. Top: Heatmap showing correlation between each pairof cells. Bottom: Heatmap showing the level of expression of each genein each cell. Cells and genes are ordered via bidirectional hierarchicalclustering.

FIGS. 52A-F illustrate data plotted in principal component space. Colorindicates log(number of unique transcript molecules detected) for theparticular gene. FIG. 52A. Genes that appear to be expressed by a largerproportion of cells upon stimulation by CMV peptide pool. FIG. 52B.Genes that are enriched in one branch of cells. These genes are alsoknown to be associated with naive and central memory CD8+ T cells. FIG.52C. Genes that are enriched in the other branch of cells. These genesare known to be associated with effector and effector memory CD8+ Tcells. FIG. 52D. Granzyme K expressing cells occupy a region between thenaive/central memory and effector/effector memory cells on the PC space.FIG. 52E. HLA-DRA expressing cells constitute a special subset. FIG.52F. Genes that are expressed in both branches of cells.

FIG. 53. Same as FIG. 50B, except the data represents those of theunstimulated controls. None of the cells in Donor 1's sample expressedIFNG, while one cell in Donor 2's sample expressed IFNG yet with overalllow expression across the entire gene panel (rank 1069). Color scale isadjusted to match that of the respective graph for the stimulatedsample.

FIG. 54. Heatmaps showing the heterogeneous expression of the gene panelin cells that express gamma interferon (IFNG) in CMV stimulated CD8+ Tcells of Donors 1 and 2. Also shown is the cell that carries most totaltranscripts detected in Donor 2. This particular cell does not expressIFNG but expresses strongly IL6, IL1B and CCL4. The cells and genes areordered by bidirectional hierarchical clustering based on correlation.Cell ID refers to the rank in total number of detected transcripts ofthe gene panel, and are indicated in the PCA plots in FIG. 50.

FIG. 55. Amplification scheme. The first PCR amplifies moleculesattached to the bead using a gene specific primer and a primer againstthe universal Illumina sequencing primer 1 sequence. The second PCRamplifies the first PCR products using a nested gene specific primerflanked by Illumina sequencing primer 2 sequence, and a primer againstthe universal Illumina sequencing primer 1 sequence. The third PCR addsP5 and P7 and sample index to turn PCR products into Illumina sequencinglibrary. 150 bp×2 sequencing reveals the cell label and molecule labelon read 1, the gene on read 2, and the sample index on index 1 read.

FIG. 56 depicts a schematic of a workflow for analyzing molecules from asample. FIG. 56 discloses “AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA” as SEQ IDNO: 831.

FIG. 57 depicts a schematic of a workflow for analyzing molecules from asample.

FIG. 58A-B depict agarose gels of PCR products.

FIG. 59 depicts a plot of sequencing reads for a plurality of genes.

FIG. 60A-D depicts plots of the reads observed per label detected (RPLD)for Lys, Phe, Thr, and Dap spike-in controls, respectively. FIG. 60Edepicts a plot of Reads versus Input.

FIG. 61 depicts a plot of the reads observed per label detected (RPLD)for various genes.

FIG. 62 depicts a plot of the reads observed per label detected (RPLD)for various genes.

FIG. 63 depicts a plot of total reads (labels) versus rpld for variousgenes.

FIG. 64 depicts a plot of RPKM for undetected genes.

FIG. 65 depicts a schematic for the synthesis of molecular barcodes.FIG. 65 discloses “1001” as SEQ ID NO: 832 and “1003” as SEQ ID NO: 833and “1005” as SEQ ID NOS 832 and 833, respectively, in order ofappearance.

FIG. 66A-C depict schematics for the synthesis of molecular barcodes.FIG. 66A discloses “1121” as SEQ ID NO: 834, “1127” as SEQ ID NO: 835,“1128” as SEQ ID NO: 836 and “1129” as SEQ ID NO: 837. FIG. 66Bdiscloses “1150” as SEQ ID NO: 838, “1159” as SEQ ID NO: 839 and “1158”as SEQ ID NO: 840. FIG. 66C discloses “1170” as SEQ ID NO: 841, “1176”as SEQ ID NO: 842 and “1177” as SEQ ID NO: 843.

FIG. 67 shows a schematic of a workflow for stochastically labelingnucleic acids. FIG. 67 discloses “AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA” asSEQ ID NO: 831.

FIG. 68 is a schematic of a workflow for stochastically labeling nucleicacids. FIG. 68 discloses “AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA” as SEQ ID NO:831.

FIG. 69 illustrates a mechanical fixture within which microwell arraysubstrates may be clamped, thereby forming a reaction chamber or wellinto which samples and reagents may be pipetted for performingmultiplexed, single cell stochastic labeling/molecular indexingexperiments. Upper: exploded view showing the upper and lower parts ofthe fixture and an elastomeric gasket for forming a leak-proof seal withthe microwell array substrate. Lower: exploded side-view of the fixture.

FIG. 70 illustrates a mechanical fixture which creates two reactionchambers or wells when a microwell array substrate is clamped within thefixture.

FIG. 71 illustrates two examples of elastomeric (e.g.,polydimethylsiloxane) gaskets for use with the mechanical fixturesillustrated in FIGS. 69 and 70. The elastomeric gaskets provide for aleak-proof seal with the microwell array substrate to create a reagentwell around the microwell array. The gaskets may contain one (upper),two (lower), or more openings for creating reagent wells.

FIG. 72 depicts one embodiment of a cartridge within which a microwellarray is packaged. Left: An exploded view of the cartridge illustrating(from bottom to top) the microwell array substrate, a gasket thatdefines the flow cell or array chamber, a reagent and/or waste reservoircomponent for defining compartments to contain pre-loaded assay reagentsor store spent reagents, and a cover for sealing the reagent and wastereservoirs and defining the sample inlet and outlet ports. Right: Anassembled view of one embodiment of the cartridge design illustratingrelief for bringing an external magnet into close proximity with themicrowell array.

FIG. 73 depicts one embodiment of a cartridge designed to includeonboard assay reagents with the packaged microwell array.

FIG. 74 provides a schematic illustration of an instrument system forperforming multiplexed, single cell stochastic labeling/molecularindexing assay. The instrument system may provide a variety of controland analysis capabilities, and may be packaged as individual modules oras a fully integrated system. Microwell arrays may be integrated withflow cells that are either a fixed component of the system or areremovable, or may be packaged within removable cartridges that furthercomprise pre-loaded assay reagent reservoirs and other functionality.

FIG. 75 illustrates one embodiment of the process steps to be performedby an automated system for performing multiplexed, single cellstochastic labeling/molecular indexing assays.

FIG. 76 illustrates one embodiment of a computer system or processor forproviding instrument control and data analysis capabilities for theassay system presently disclosed.

FIG. 77 shows a block diagram illustrating one example of a computersystem architecture that can be used in connection with exampleembodiments of the assay systems of the present disclosure.

FIG. 78 depicts a diagram showing a network with a plurality of computersystems, cell phones, personal data assistants, and Network AttachedStorage (NAS), that can be used with example embodiments of the assaysystems of the present disclosure.

FIG. 79 depicts a block diagram of a multiprocessor computer system thatcan be used with example embodiments of the assay systems of the presentdisclosure.

FIG. 80 depicts a diagram of analysis of a test sample and communicationof test result obtained from the test sample via a communication media.

DETAILED DESCRIPTION

Disclosed herein are methods, kits, and compositions for analyzingmolecules in a plurality of samples. Generally, the methods, kits, andcompositions comprise (a) stochastically labeling molecules in two ormore samples with molecular barcodes to produce labeled molecules; and(b) detecting the labeled molecules. The molecular barcodes may compriseone or more target specific regions, label regions, sample indexregions, universal PCR regions, adaptors, linkers, or a combinationthereof. The labeled molecules may comprise a) a molecule region; b) asample index region; and c) a label region. The molecule region maycomprise at least a portion of the molecule from the molecular barcodewas originally attached to. The molecule region may comprise a fragmentof the molecule from the molecular barcode was originally attached to.The sample index region may be used to determine the source of themolecule region. The sample index region may be used to determine fromwhich sample the molecule region originated from. The sample indexregion may be used to differentiate molecule regions from two or moredifferent samples. The label region may be used to confer a uniqueidentity to identical molecule regions originating from the same source.The label region may be used to confer a unique identity to identicalmolecule regions originating from the same sample.

The method for analyzing molecules in a plurality of samples maycomprise: a) producing a plurality of sample-tagged nucleic acids by: i)contacting a first sample comprising a plurality of nucleic acids with aplurality of first sample tags to produce a plurality of firstsample-tagged nucleic acids; and ii) contacting a second samplecomprising a plurality of nucleic acids with a plurality of secondsample tags to produce a plurality of second sample-tagged nucleicacids, wherein the plurality of second sample tags are different fromthe first sample tags; b) contacting the plurality of sample-taggednucleic acids with a plurality of molecular identifier labels to producea plurality of labeled nucleic acids; and c) detecting at least aportion of the labeled nucleic acids, thereby determining a count of aplurality of nucleic acids in a plurality of samples. The plurality ofsamples may comprise a single cell.

Alternatively, the method for analyzing molecules in a plurality ofsamples may comprise: a) producing a plurality of labeled nucleic acidscomprising: i) contacting a first sample with a first plurality ofsample tags, wherein the first plurality of sample tags comprisesidentical nucleic acid sequences; ii) contacting the first sample with afirst plurality of molecular identifier labels may comprise differentnucleic acid sequences, wherein contacting the first sample with thefirst plurality of sample tags or first plurality of molecularidentifier labels occurs simultaneously or sequentially to produce aplurality of first-labeled nucleic acids; iii) contacting a secondsample with a second plurality of sample tags, wherein the secondplurality of sample tags may comprise identical nucleic acid sequences;iv) contacting the second sample with a second plurality of molecularidentifier labels may comprise different nucleic acid sequences, whereincontacting the second sample with the second plurality of sample tags orsecond plurality of molecular identifier labels occurs simultaneously orsequentially to produce a plurality of second-labeled nucleic acids,wherein the plurality of labeled nucleic acids may comprise theplurality of first-labeled nucleic acids and the second-labeled nucleicacids; and b) determining a number of different labeled nucleic acids,thereby determining a count of a plurality of nucleic acids in aplurality of samples.

The method for analyzing molecules in a plurality of samples maycomprise: a) contacting a plurality of samples may comprise two or moredifferent nucleic acids with a plurality of sample tags and a pluralityof molecular identifier labels to produce a plurality of labeled nucleicacids, wherein: i) the plurality of labeled nucleic acids may comprisetwo or more nucleic acids attached to two or more sample tags and two ormore molecular identifier labels; ii) the sample tags attached tonucleic acids from a first sample of the plurality of samples aredifferent from the sample tags attached to nucleic acid molecules from asecond sample of the plurality of samples; and iii) two or moreidentical nucleic acids in the same sample are attached to two or moredifferent molecular identifier labels; and b) detecting at least aportion of the labeled nucleic acids, thereby determining a count of twoor more different nucleic acids in the plurality of samples.

FIG. 56 depicts an exemplary workflow for the quantification of RNAmolecules in a sample. As shown in Step 1 of FIG. 56, RNA molecules(110) may be reverse transcribed to produce cDNA molecules (105) by thestochastic hybridization of a set of molecular identifier labels (115)to the polyA tail region of the RNA molecules. The molecular identifierlabels (115) may comprise an oligodT region (120), label region (125),and universal PCR region (130). The set of molecular identifier labelsmay contain 960 different types of label regions. As shown in Step 2 ofFIG. 56, the labeled cDNA molecules (170) may be purified to removeexcess molecular identifier labels (115). Purification may compriseAmpure bead purification. As shown in Step 3 of FIG. 56, the labeledcDNA molecules (170) may be amplified to produce a labeled amplicon(180). Amplification may comprise multiplex PCR amplification.Amplification may comprise a multiplex PCR amplification with 96multiplex primers in a single reaction volume. Amplification maycomprise a custom primer (135) and a universal primer (140). The customprimer (135) may hybridize to a region within the cDNA (105) portion ofthe labeled cDNA molecule (170). The universal primer (140) mayhybridize to the universal PCR region (130) of the labeled cDNA molecule(170). As shown in Step 4, the labeled amplicons (180) may be furtheramplified by nested PCR. The nested PCR may comprise multiplex PCR with96 multiplex primers in a single reaction volume. Nested PCR maycomprise a custom primer (145) and a universal primer (140). The customprimer (135) may hybridize to a region within the cDNA (105) portion ofthe labeled amplicon (180). The universal primer (140) may hybridize tothe universal PCR region (130) of the labeled amplicon (180). As shownin Step 5, one or more adaptors (150, 155) may be attached to thelabeled amplicon (180) to produce an adaptor-labeled amplicon (190). Theone or more adaptors may be attached to the labeled amplicon (180) vialigation. As shown in Step 6, the one or more adaptors (150, 155) may beused to conduct one or more additional assays on the adaptor-labeledamplicon (190). The one or more adaptors (150, 155) may be hybridized toone or more primers (160, 165). The one or more primers (160, 165) bePCR amplification primers. The one or more primers (160, 165) may besequencing primers. The one or more adaptors (150, 155) may be used forfurther amplification of the adaptor-labeled amplicons. The one or moreadaptors (150, 155) may be used for sequencing the adaptor-labeledamplicon.

FIG. 57 depicts an exemplary schematic of a workflow for analyzingnucleic acids from two or more samples. As shown in FIG. 57, a methodfor analyzing nucleic acids from two or more samples may compriseselecting two or more genes for analysis and designing custom primersbased on the selected genes (210). The method may further comprisesupplementing one or more samples comprising nucleic acids (e.g., RNA)with one or more spike-in controls (220). The nucleic acids in thesample may be amplified by multiplex RT-PCR (230) with molecularbarcodes (or sample tags or molecular identifier labels) and the customprimers to produce labeled amplicons. The labeled amplicons may furthertreated with one or more sequencing adaptors to produce adaptor labeledamplicons (240). The adaptor labeled amplicons can be analyzed (250). Asshown in FIG. 57, analysis of the labeled amplicons (250) may compriseone or more of (1) detection of a universal PCR primer seq, polyA and/ormolecular barcode (or sample tag, molecular identifier label); (2) mapread on the end of the adaptor labeled amplicons (e.g., 96 genes andspike-in controls) that is not attached to the adaptor and/or barcode(e.g., molecular barcode, sample tag, molecular identifier label); and(3) count and/or summarize the number of different adaptor labeledamplicons.

FIG. 67 shows a schematic of a workflow for stochastically labelingnucleic acids with molecular barcodes (1220). As shown in step 1 of FIG.67, RNA molecules may be stochastically labeled with a set of molecularbarcodes (1220). The molecular barcodes (1220) may comprise a targetbinding region (1221), label region (1222), sample index region (1223)and universal PCR region (1224). In some instances, the target bindingregion comprises an oligodT sequence that hybridizes to a polyA sequencein the RNA molecules. The label region (1222) may contain a uniquesequence that may be used to distinguish two or more different molecularbarcodes. When the molecular barcode hybridizes to an RNA molecule, thelabel region may be used to confer a unique identity to identical RNAmolecules. The sample index region (1223) may be identical for a set ofmolecular barcodes. The sample index region (1223) may be used todistinguish labeled nucleic acids from different samples. The universalPCR region (1224) may serve as a primer binding site for amplificationof the labeled molecules. Once the RNA molecules are labeled with themolecular barcodes, the RNA molecules may be reverse transcribed toproduce labeled cDNA molecules (1230) containing a cDNA copy of the RNAmolecule (1210) and the molecular barcode (1220).

As shown in Step 2 of FIG. 67, excess oligos (e.g., molecular barcodes)may be removed by Ampure bead purification. As shown in Step 3 of FIG.67, the labeled cDNA molecules may be amplified by multiplex PCR.Multiplex PCR of the labeled cDNA molecules may be performed by using afirst set of forward primers (F1, 1235 in FIG. 67) and universal primers(1240) in a single reaction volume to produce labeled amplicons (1245).As shown in Step 4 of FIG. 67, the labeled amplicons may be furtheramplified by multiplex PCR using nested primers. Nested primeramplification of the labeled amplicons may be performed by using asecond set of forward primers (F2, 1250 in FIG. 67) and universalprimers (1240) in a single reaction volume to produce labeled nested PCRamplicons. In some instances, the F2 primers (1250) contain an adaptor(1251) and a target binding region (1252). The target binding region(1252) of the F2 primers may hybridize to the labeled amplicons and mayprime amplification of the labeled amplicons. The adaptor (1251) and theuniversal PCR region (1224) of the nested PCR amplicons may be used inthe sequencing of the labeled nested PCR amplicons. The amplicons may besequenced by MiSeq. Alternatively, the amplicons may be sequenced byHiSeq.

FIG. 68 shows a schematic of a workflow for stochastically labelingnucleic acids. As shown in Step 1 of FIG. 68, RNA molecules (1305) maybe stochastically labeled with a set molecular barcodes (1320). Themolecular barcodes may comprise a target binding region (1321), labelregion (1322), and universal PCR region (1323). Once the molecularbarcodes are attached to the RNA molecules, the RNA molecules (1305) maybe reverse transcribed to produce labeled cDNA molecules (1325)comprising a cDNA copy of the RNA molecule (1310) and the molecularbarcode (1320). As shown in Step 2 of FIG. 68, the labeled cDNAmolecules may be purified by Ampure bead purification to remove excessoligos (e.g., molecular barcodes). As shown in Step 3 of FIG. 68, thelabeled amplicons may be amplified by multiplex PCR. Multiplex PCR ofthe labeled cDNA molecules may be performed by using a first set offorward primers (F1, 1330 in FIG. 68) and universal primers (1335) in asingle reaction volume to produce labeled amplicons (1360). As shown inStep 4 of FIG. 67, the labeled amplicons may be further amplified bymultiplex PCR using nested primers. Nested primer amplification of thelabeled amplicons may be performed by using a second set of forwardprimers (F2, 1340 in FIG. 68) and sample index primers (1350) in asingle reaction volume to produce labeled nested PCR amplicons. In someinstances, the F2 primers (1340) contain an adaptor (1341) and a targetbinding region (1342). The target binding region (1342) of the F2primers may hybridize to the labeled amplicons and may primeamplification of the labeled amplicons. The sample index primers (1350)may comprise a universal primer region (1351), sample index region(1352), and adaptor region (1353). As shown in Step 4 of FIG. 68, theuniversal primer region (1351) of the sample index primer may hybridizeto the universal PCR region of the labeled amplicons. The sample indexregion (1352) of the sample index primer may be used to distinguish twoor more samples. The adaptor regions (1341, 1353) may be used tosequence the labeled nested PCR amplicons. The amplicons may besequenced by MiSeq. Alternatively, the amplicons may be sequenced byHiSeq.

Further disclosed herein are methods of producing one or more libraries.The one or more libraries may comprise a plurality of labeled molecules.The one or more libraries may comprise a plurality of labeled amplicons.The one or more libraries may comprise a plurality of enriched moleculesor a derivative thereof (e.g., labeled molecules, labeled amplicons).Generally, the method of producing one or more libraries comprises (a)stochastically labeling a plurality of molecules from two or moresamples to produce a plurality of labeled molecules, wherein the labeledmolecules comprise a molecule region, a sample index region, and labelregion; and (b) producing one or more libraries from the plurality oflabeled molecules, wherein (i) the one or more libraries comprise two ormore different labeled molecules, (ii) the two or more different labeledmolecules differ by the molecule region, sample index region, labelregion, or a combination thereof.

The method for producing one or more libraries may comprise: a)producing a plurality of sample-tagged nucleic acids by: i) contacting afirst sample comprising a plurality of nucleic acids with a plurality offirst sample tags to produce a plurality of first sample-tagged nucleicacids; and ii) contacting a second sample comprising a plurality ofnucleic acids with a plurality of second sample tags to produce aplurality of second sample-tagged nucleic acids, wherein the pluralityof first sample tags are different from the second sample tags; and b)contacting the plurality of sample-tagged nucleic acids with a pluralityof molecular identifier labels to produce a plurality of labeled nucleicacids, thereby producing a labeled nucleic acid library.

The contacting to a sample can be random or non-random. For example, thecontacting of a sample with sample tags can be a random or non-randomcontacting. In some embodiments, the sample is contacted with sampletags randomly. In some embodiments, the sample is contacted with sampletags non-randomly. The contacting to a plurality of nucleic acids can berandom or non-random. For example, the contacting of a plurality ofnucleic acids with sample tags can be a random or non-random contacting.In some embodiments, the plurality of nucleic acids is contacted withsample tags randomly. In some embodiments, the plurality of nucleicacids is contacted with sample tags non-randomly.

Further disclosed herein are methods of producing one or more sets oflabeled beads. The method of producing the one or more sets of labeledbeads may comprise attaching one or more nucleic acids to one or morebeads, thereby producing one or more sets of labeled beads. The one ormore nucleic acids may comprise one or more molecular barcodes. The oneor more nucleic acids may comprise one or more sample tags. The one ormore nucleic acids may comprise one or more molecular identifier labels.The one or more nucleic acids may comprise a) a primer region; b) asample index region; and c) a linker or adaptor region. The one or morenucleic acids may comprise a) a primer region; b) a label region; and c)a linker or adaptor region. The one or more nucleic acids may comprisea) a sample index region; and b) a label region. The one or more nucleicacids may further comprise a primer region. The one or more nucleicacids may further comprise a target specific region. The one or morenucleic acids may further comprise a linker region. The one or morenucleic acids may further comprise an adaptor region. The one or morenucleic acids may further comprise a sample index region. The one ormore nucleic acids may further comprise a label region.

Further disclosed herein are methods for selecting one or more customprimers. The method of selecting a custom primer for analyzing moleculesin a plurality of samples may comprise: a) a first pass, wherein primerschosen may comprise: i) no more than three sequential guanines, no morethan three sequential cytosines, no more than four sequential adenines,and no more than four sequential thymines; ii) at least 3, 4, 5, or 6nucleotides that are guanines or cytosines; and iii) a sequence thatdoes not easily form a hairpin structure; b) a second pass, comprising:i) a first round of choosing a plurality of sequences that have highcoverage of all transcripts; and ii) one or more subsequent rounds,selecting a sequence that has the highest coverage of remainingtranscripts and a complementary score with other chosen sequences nomore than 4; and c) adding sequences to a picked set until coveragesaturates or total number of customer primers is less than or equal toabout 96.

Further disclosed herein are kits for use in analyzing two or moremolecules from two or more samples. The kit may comprise (a) a firstcontainer comprising a first set of molecular barcodes, wherein (i) amolecular barcode of the first set of molecular barcodes comprise asample index region and a label region; (ii) the sample index region oftwo or more barcodes of the first set of molecular barcodes are thesame; and (iii) the label region of two or more barcodes of the firstset of molecular barcodes are different; and (b) a second containercomprising a second set of molecular barcodes, wherein (i) a molecularbarcode of the second set of molecular barcodes comprise a sample indexregion and a label region; (ii) the sample index region of two or morebarcodes of the second set of molecular barcodes are the same; (iii) thelabel region of two or more barcodes of the second set of molecularbarcodes are different; (iv) the sample index region of the barcodes ofthe second set of molecular barcodes are different from the sample indexregion of the barcodes of the first set of molecular barcodes; and (v)the label region of two or more barcodes of the second set of molecularbarcodes are identical to the label region of two or more barcodes ofthe first set of molecular barcodes.

Alternatively, the kit comprises: a) a plurality of beads, wherein oneor more beads of the plurality of beads may comprise at least one of aplurality of nucleic acids, wherein at least one of a plurality nucleicacids may comprise: i) at least one primer sequence, wherein the primersequence of at least one of the plurality of nucleic acids is the samefor the plurality of beads; ii) a bead-specific sequence, wherein thebead-specific sequence of any one of the plurality of nucleic acids isthe same, and wherein the bead-specific sequence is different for anyone of the plurality of beads; and iii) a stochastic sequence, whereinthe stochastic sequence is different for any one of the plurality ofnucleic acids; b) a primer may comprise a sequence complementary to theprimer sequence; and c) one or more amplification agents suitable fornucleic acid amplification.

Alternatively, the kit comprises: a) a first container comprising afirst set of sample tags, wherein (i) a sample tag of the first set ofsample tags comprises a sample index region; and (ii) the sample indexregions of the sample tags of the first set of sample tags are at leastabout 80% identical; and b) a second container comprising a first set ofmolecular identifier labels, wherein (i) a molecular identifier label ofthe first set of molecular identifier labels comprises a label region;and (ii) at least about 30% of the label regions of the total molecularidentifier labels of the first set of molecular identifier labels aredifferent

Before the present methods, kits and compositions are described ingreater detail, it is to be understood that this invention is notlimited to particular method, kit or composition described, as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims. Examples are put forth soas to provide those of ordinary skill in the art with a completedisclosure and description of how to make and use the present invention,and are not intended to limit the scope of what the inventors regard astheir invention nor are they intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Methods, kits and compositions are provided for stochastic labeling ofnucleic acids in a plurality of samples or in a complex nucleic acidpreparation. These methods, kits and compositions find use in unravelingmechanisms of cellular response, differentiation or signal transductionand in performing a wide variety of clinical measurements. These andother objects, advantages, and features of the invention will becomeapparent to those persons skilled in the art upon reading the details ofthe methods, kits and compositions as more fully described below.

The methods disclosed herein comprise attaching one or more molecularbarcodes, sample tags, and/or molecular identifier labels to two or moremolecules from two or more samples. The molecular barcodes, sample tagsand/or molecular identifier labels may comprise one or moreoligonucleotides. In some instances, attachment of molecular barcodes,sample tags, and/or molecular identifier labels to the moleculescomprises stochastic labeling of the molecules. Methods forstochastically labeling molecules may be found, for example, in U.S.Ser. Nos. 12/969,581 and 13/327,526. Generally, the stochastic labelingmethod comprises the random attachment of a plurality of the tag andlabel oligonucleotides to one or more molecules. The molecular barcodes,sample tags, and/or molecular identifier labels are provided in excessof the one or more molecules to be labeled. In stochastic labeling, eachindividual molecule to be labeled has an individual probability ofattaching to the plurality of the molecular barcodes, sample tags,and/or molecular identifier labels. The probability of each individualmolecule to be labeled attaching to a particular molecular barcodes,sample tags, and/or molecular identifier labels may be about the same asany other individual molecule to be labeled. Accordingly, in someinstances, the probability of any of the molecules in a sample findingany of the tags and labels is assumed to be equal, an assumption thatmay be used in mathematical calculations to estimate the number ofmolecules in the sample. In some circumstances the probability ofattaching may be manipulated by, for example electing tags and labelswith different properties that would increase or decrease the bindingefficiency of that molecular barcodes, sample tags, and/or molecularidentifier labels with an individual molecule. The tags and labels mayalso be varied in numbers to alter the probability that a particularmolecular barcodes, sample tags, and/or molecular identifier labels willfind a binding partner during the stochastic labeling. For example, onelabel is overrepresented in a pool of labels, thereby increasing thechances that the overrepresented label finds at least one bindingpartner.

The methods disclosed herein may further comprise combining two or moresamples. The methods disclosed herein may further comprise combining oneor more molecules from two or more samples. For example, the methodsdisclosed herein comprise combining a first sample and a second sample.The two or more samples may be combined after conducting one or morestochastic labeling procedures. The two or more samples may be combinedafter attachment of one or more sets of molecular barcodes to two ormore molecules from the two or more samples. The two or more samples maybe combined after attachment of one or more sets of sample tags to twoor more molecules from the two or more samples. The two or more samplesmay be combined after attachment of one or more sets of molecularidentifier labels to two or more molecules from the two or more samples.For example, the first and second samples are combined prior to contactwith the plurality of molecular identifier labels.

Alternatively, the two or more samples may be combined prior toconducting one or more stochastic labeling procedures. The two or moresamples may be combined prior to attachment of one or more sets ofmolecular barcodes to two or more molecules from the two or moresamples. The two or more samples may be combined prior to attachment ofone or more sets of sample tags to two or more molecules from the two ormore samples. The two or more samples may be combined prior toattachment of one or more sets of molecular identifier labels to two ormore molecules from the two or more samples.

The two or more samples may be combined after conducting one or moreassays on two or more molecules or derivatives thereof (e.g., labeledmolecules, amplicons) from the two or more samples. The one or moreassays may comprise one or more amplification reactions. The one or moreassays may comprise one or more enrichment assays. The one or moreassays may comprise one or more detection assays. For example, the firstand second samples are combined after detecting the labeled nucleicacids.

The two or more samples may be combined prior to conducting one or moreassays on two or more molecules or derivatives thereof (e.g., labeledmolecules, amplicons) from the two or more samples. The one or moreassays may comprise one or more amplification reactions. The one or moreassays may comprise one or more enrichment assays. The one or moreassays may comprise one or more detection assays. For example, the firstand second samples are combined prior to detecting the labeled nucleicacids.

Supports

The present disclosure comprises compositions and methods for multiplexsequence analysis from single cells. The methods and compositions of thepresent disclosure provide for the use of solid supports. In someinstances, the methods, kits, and compositions disclosed herein comprisea support.

The terms “support”, “solid support”, “semi-solid support”, and“substrate” may be used interchangeably and refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. A supportmay refer to any surface that is transferable from solution to solutionor forms a structure for conducting oligonucleotide-based assays. Thesupport or substrate may be a solid support. Alternatively, the supportis a non-solid support. A support may refer to an insoluble,semi-soluble, or insoluble material. A support may be referred to as“functionalized” when it includes a linker, a scaffold, a buildingblock, or other reactive moiety attached thereto, whereas a solidsupport may be “nonfunctionalized” when it lack such a reactive moietyattached thereto. The support may be employed free in solution, such asin a microtiter well format; in a flow-through format, such as in acolumn; or in a dipstick.

The support or substrate may comprise a membrane, paper, plastic, coatedsurface, flat surface, glass, slide, chip, or any combination thereof.In many embodiments, at least one surface of the support may besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different compounds with, forexample, wells, raised regions, pins, etched trenches, or the like.According to other embodiments, the solid support(s) may take the formof resins, gels, microspheres, or other geometric configurations.Alternatively, the solid support(s) comprises silica chips,microparticles, nanoparticles, plates, and arrays. Solid supports mayinclude beads (e.g., silica gel, controlled pore glass, magnetic beads,Dynabeads, Wang resin; Merrifield resin, Sephadex/Sepharose beads,cellulose beads, polystyrene beads etc.), capillaries, flat supportssuch as glass fiber filters, glass surfaces, metal surfaces (steel, goldsilver, aluminum, silicon and copper), glass supports, plastic supports,silicon supports, chips, filters, membranes, microwell plates, slides,or the like. plastic materials including multiwell plates or membranes(e.g., formed of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), wafers, combs, pins or needles (e.g., arraysof pins suitable for combinatorial synthesis or analysis) or beads in anarray of pits or nanoliter wells of flat surfaces such as wafers (e.g.,silicon wafers), wafers with pits with or without filter bottoms.

Methods and techniques applicable to polymer (including protein) arraysynthesis have been described in U.S. Patent Pub. No. 20050074787, WO00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633,5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074,5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695,5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101,5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956,6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and6,428,752, in PCT Publication No. WO 99/36760 and WO 01/58593, which areall incorporated herein by reference in their entirety for all purposes.Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but many of the same techniques may be applied topolypeptide arrays. Additional exemplary substrates are disclosed inU.S. Pat. No. 5,744,305 and US Patent Pub. Nos. 20090149340 and20080038559.

The attachment of the labeled nucleic acids to the support may compriseamine-thiol crosslinking, maleimide crosslinking, N-hydroxysuccinimideor N-hydroxysulfosuccinimide, Zenon or SiteClick. Attaching the labelednucleic acids to the support may comprise attaching biotin to theplurality of labeled nucleic acids and coating the one or more beadswith streptavadin.

In some instances, a solid support may comprise a molecular scaffold.Exemplary molecular scaffolds may include antibodies, antigens, affinityreagents, polypeptides, nucleic acids, cellular organelles, and thelike. Molecular scaffolds may be linked together (e.g., a solid supportmay comprise a plurality of connected molecular scaffolds). Molecularscaffolds may be linked together by an amino acid linker, a nucleic acidlinker, a small molecule linkage (e.g., biotin and avidin), and/or amatrix linkage (e.g., PEG or glycerol). Linkages may be non-covalent.Linkages may be covalent. In some instances, molecular scaffolds may notbe linked. A plurality of individual molecular scaffolds may be used inthe methods of the disclosure.

In some instances a support may comprise a nanoparticle. Thenanoparticle may be a nickel, gold, silver, carbon, copper, silicate,platinum cobalt, zinc oxide, silicon dioxide crystalline, and/or silvernanoparticle. Alternatively, or additionally, the nanoparticle may be agold nanoparticle embedded in a porous manganese oxide. The nanoparticlemay be an iron nanoparticle. The nanoparticle may be a nanotetrapodstudded with nanoparticles of carbon.

A support may comprise a polymer. A polymer may comprise a matrix. Amatrix may further comprise one or more beads. A polymer may comprisePEG, glycerol, polysaccharide, or a combination thereof. A polymer maybe a plastic, rubber, nylon, silicone, neoprene, and/or polystyrene. Apolymer may be a natural polymer. Examples of natural polymers include,but are not limited to, shellac, amber, wool, silk, cellulose, andnatural rubber. A polymer may be a synthetic polymer. Examples ofsynthetic polymers include, but are not limited to, synthetic rubber,phenol formaldehyde resin (or Bakelite), neoprene, nylon, polyvinylchloride (PVC or vinyl), polystyrene, polyethylene, polypropylene,polyacrylonitrile, PVB, and silicone.

A support may be a semi-solid support. A support may comprise a gel(e.g., a hydrogel). The terms “hydrogel”, “gel” and the like, are usedinterchangeably herein and may refer to a material which is not areadily flowable liquid and not a solid but a gel which gel is comprisedof from 0.5% or more and preferably less than 40% by weight of gelforming solute material and from 95% or less and preferably more than55% water. The gels of the invention may be formed by the use of asolute which is preferably a synthetic solute (but could be a naturalsolute, e.g., for forming gelatin) which forms interconnected cellswhich binds to, entrap, absorb and/or otherwise hold water and therebycreate a gel in combination with water, where water includes bound andunbound water. The gel may be the basic structure of the hydrogel patchof the invention will include additional components beyond the gelforming solute material and water such as an enzyme and a salt whichcomponents are further described herein. The gel may be a polymer gel.

A solid support may comprise a structured nanostructure. For example,the structured nanostructure may comprise capture containers (e.g., aminiaturized honeycomb) which may comprise the oligonucleotides tocapture the cell and/or contents of the cell. In some instances,structured nanostructures may not need the addition of exogenousreagents.

In some instances, the support comprises a bead. A bead may encompassany type of solid or hollow sphere, ball, bearing, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial onto which a nucleic acid may be immobilized (e.g., covalentlyor non-covalently). A bead may comprise nylon string or strings. A beadmay be spherical in shape. A bead may be non-spherical in shape. Beadsmay be unpolished or, if polished, the polished bead may be roughenedbefore treating, (e.g., with an alkylating agent). A bead may comprise adiscrete particle that may be spherical (e.g., microspheres) or have anirregular shape. Beads may comprise a variety of materials including,but not limited to, paramagnetic materials, ceramic, plastic, glass,polystyrene, methylstyrene, acrylic polymers, titanium, latex,sepharose, cellulose, nylon and the like. A bead may be attached to orembedded into one or more supports. A bead may be attached to a gel orhydrogel. A bead may be embedded into a gel or hydrogel. A bead may beattached to a matrix. A bead may be embedded into a matrix. A bead maybe attached to a polymer. A bead may be embedded into a polymer. Thespatial position of a bead within the support (e.g., gel, matrix,scaffold, or polymer) may be identified using the oligonucleotidepresent on the bead which serves as a location address. Examples ofbeads include, but are not limited to, streptavidin beads, agarosebeads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugatedbeads (e.g., anti-immunoglobulin microbead), protein A conjugated beads,protein G conjugated beads, protein A/G conjugated beads, protein Lconjugated beads, oligodT conjugated beads, silica beads, silica-likebeads, anti-biotin microbead, anti-fluorochrome microbead, and BcMag™Carboxy-Terminated Magnetic Beads. The diameter of the beads may beabout 5 μm, 10 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm. Abead may refer to any three dimensional structure that may provide anincreased surface area for immobilization of biological particles andmacromolecules, such as DNA and RNA.

A support may be porous. A support may be permeable or semi-permeable. Asupport may be solid. A support may be semi-solid. A support may bemalleable. A support may be flexible. In some instances, a support maybe molded into a shape. For example, a support may be placed over anobject and the support may take the shape of the object. In someinstances, the support is placed over an organ and takes the shape ofthe organ. In some instances, the support is produced by 3D-printing.

The support (e.g., beads, nanoparticles) may be at least about 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 100, 500, 1000, or 2000 or moremicrometers in diameter. The solid supports (e.g., beads) may be at mostabout 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 100, 500, 1000,or 2000 or more micrometers in diameter. The diameter of the bead may beabout 20 microns.

In some instances, a solid support comprises a dendrimer. A dendrimermay be smaller than a bead. A dendrimer may be subcellular. A dendrimermay be less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1micron in diameter. A dendrimer may be less than 0.09, 0.08, 0.07, 0.06,0.05, 0.04, 0.03, or 0.01 micron in diameter A dendrimer may comprisethree major portions, a core, an inner shell, and an outer shell. Adendrimer may be synthesized to have different functionality in each ofthese portions. The different functionality of the portions of thedendrimer may control properties such as solubility, thermal stability,and attachment of compounds for particular applications. A dendrimer maybe synthetically processed. A dendrimer may be synthesized by divergentsynthesis. Divergent synthesis may comprise assembling a dendrimer froma multifunctional core, which is extended outward by a series ofreactions. Divergent synthesis may comprise a series of Michaelreactions. Alternatively, a dendrimer may be synthesized by convergentsynthesis. Convergent synthesis may comprise building dendrimers fromsmall molecules that end up at the surface of the sphere, and reactionsmay proceed inward and are eventually attached to a core. Dendrimers mayalso be prepared by click chemistry. Click chemistry may compriseDiels-Alder reactions, thiol-yne reactions, azide-alkyne reactions, or acombination thereof. Examples of dendrimers include, but are not limitedto, poly(amidoamine) (PAMAM) dendrimer, PEG-core denderimer, phosphorousdendrimer, polypropylenimine dendrimer, and polylysine dendrimer. Adendrimer may be a chiral dendrimer. Alternatively, a dendrimer may bean achiral dendrimer.

A solid support may comprise a portion of a dendrimer. The portion ofthe dendrimer may comprise a dendron. A dendron may comprisemonodisperse wedge-shaped dendrimer sections with multiple terminalgroups and a single reaction function at the focal point. A solidsupport may comprise a polyester dendrom. Examples of dendrons include,but are not limited to, polyester-8-hydroxyl-1-acetylene bis-MPAdendron, polyester-16-hydroxyl-1-acetylene bis-MPA dendron,polyester-32-hydroxyl-1-acetylene bis-MPA dendron,polyester-8-hydroxyl-1-carboxyl bis-MPA dendron,polyester-16-hydroxyl-1-carboxyl bis-MPA dendron, andpolyester-32-hydroxyl-1-carboxyl bis-MPA dendron.

A solid support may comprise a hyberbranched polymer. A hyperbranchedpolymer may comprise polydisperse dendritic macromolecules that possessdendrimer-like properties. Often, hyberbranched polymers are prepared ina single synthetic polymerization step. The hyperbranched polymer may bebased on 2,2-bis(hydroxymethyl)propanoic acid (bis-MPA) monomer.Examples of hyperbranched polymers include, but are not limited to,hyperbranched bis-MPA polyester-16-hydroxyl, hyperbranched bis-MPApolyester-32-hydroxyl, and hyperbranched bis-MPA polyester-64-hydroxyl.

The solid support may be an array or microarray. The solid support maycomprise discrete regions. The solid support may be an addressablearray. In some instances, the array comprises a plurality of probesfixed onto a solid surface. The plurality of probes enableshybridization of the labeled-molecule and/or labeled-amplicon to thesolid surface. The plurality of probes comprises a sequence that iscomplementary to at least a portion of the labeled-molecule and/orlabeled-amplicon. In some instances, the plurality of probes comprises asequence that is complementary to at least a portion of the sample tag,molecular identifier label, nucleic acid, or a combination thereof. Inother instances, the plurality of probes comprises a sequence that iscomplementary to the junction formed by the attachment of the sample tagor molecular identifier label to the nucleic acid.

The array may comprise one or more probes. The probes may be in avariety of formats. The array may comprise a probe comprising a sequencethat is complementary to at least a portion of the target nucleic acidand a sequence that is complementary to the unique identifier region ofa sample tag or molecular identifier label, wherein the sample tag ormolecular identifier label comprises an oligonucleotide. The sequencethat is complementary to at least a portion of the target nucleic acidmay be attached to the array. The sequence that is complementary to theunique identifier region may be attached to the array. The array maycomprise a first probe comprising a sequence that is complementary to atleast a portion of the target nucleic acid and a second probe that iscomplementary to the unique identifier region. There are various ways inwhich a stochastically labeled nucleic acid may hybridize to the arrays.For example, the junction of the unique identifier region and the targetnucleic acid of the stochastically labeled nucleic acid may hybridize tothe probe on the array. There may be a gap in the regions of thestochastically labeled nucleic acid that may hybridize to the probe onthe array. Different regions of the stochastically labeled nucleic acidmay hybridize to two or more probes on the array. Thus, the array probesmay be in many different formats. The array probes may comprise asequence that is complementary to a unique identifier region, a sequencethat is complementary to the target nucleic acid, or a combinationthereof. Hybridization of the stochastically labeled nucleic acid to thearray may occur by a variety of ways. For example, two or morenucleotides of the stochastically labeled nucleic acid may hybridize toone or more probes on the array. The two or more nucleotides of thestochastically labeled nucleic acid that hybridize to the probes may beconsecutive nucleotides, non-consecutive nucleotides, or a combinationthereof. The stochastically labeled nucleic acid that is hybridized tothe probe may be detected by any method known in the art. For example,the stochastically labeled nucleic acids may be directly detected.Directly detecting the stochastically labeled nucleic acid may comprisedetection of a fluorophore, hapten, or detectable label. Thestochastically labeled molecules may be indirectly detected. Indirectdetection of the stochastically labeled nucleic acid may compriseligation or other enzymatic or non-enzymatic methods.

The array may be in a variety of formats. For example, the array may bein a 16-, 32-, 48-, 64-, 80-, 96-, 112-, 128-, 144-, 160-, 176-, 192-,208-, 224-, 240-, 256-, 272-, 288-, 304-, 320-, 336-, 352-, 368-, 384-,or 400-format. Alternatively, the array is in an 8×0.60K, 4×180K,2×400K, 1×1M format. In other instances, the array is in an 8×15K,4×44K, 2×105K, 1×244K format.

The array may comprise a single array. The single array may be on asingle substrate. Alternatively, the array is on multiple substrates.The array may comprise multiple formats. The array may comprise aplurality of arrays. The plurality of arrays may comprise two or morearrays. For example, the plurality of arrays may comprise at least about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 arrays.In some instances, at least two arrays of the plurality of arrays areidentical. Alternatively, at least two arrays of the plurality of arraysare different.

In some instances, the array comprises symmetrical chambered areas. Forexample, the array comprises 0.5×0.5 millimeters (mm), 1×1 mm, 1.5×1.5mm, 2×2 mm, 2.5×2.5 mm, 3×3 mm, 3.5×3.5 mm, 4×4 mm, 4.5×4.5 mm, 5×5 mm,5.5×5.5 mm, 6×6 mm, 6.5×6.5 mm, 7×7 mm, 7.5×7.5 mm, 8×8 mm, 8.5×8.5 mm,9×9 mm, 9.5×9.5 mm, 10×10 mm, 10.5×10.5 mm, 11×11 mm, 11.5×11.5 mm,12×12 mm, 12.5×12.5 mm, 13×13 mm, 13.5×13.5 mm, 14×14 mm, 14.5×14.5 mm,15×15 mm, 15.5×15.5 mm, 16×16 mm, 16.5×16.5 mm, 17×17 mm, 17.5×17.5 mm,18×18 mm, 18.5×18.5 mm, 19×19 mm, 19.5×19.5 mm, or 20×20 mm chamberedareas. In some instances, the array comprises 6.5×6.5 mm chamberedareas. Alternatively, the array comprises asymmetrical chambered areas.For example, the array comprises 6.5×0.5 mm, 6.5×1 mm, 6.5×1.5 mm, 6.5×2mm, 6.5×2.5 mm, 6.5×3 mm, 6.5×3.5 mm, 6.5×4 mm, 6.5×4.5 mm, 6.5×5 mm,6.5×5.5 mm, 6.5×6 mm, 6.5×6.5 mm, 6.5×7 mm, 6.5×7.5 mm, 6.5×8 mm,6.5×8.5 mm, 6.5×9 mm, 6.5×9.5 mm, 6.5×10 mm, 6.5×10.5 mm, 6.5×11 mm,6.5×11.5 mm, 6.5×12 mm, 6.5×12.5 mm, 6.5×13 mm, 6.5×13.5 mm, 6.5×14 mm,6.5×14.5 mm, 6.5×15 mm, 6.5×15.5 mm, 6.5×16 mm, 6.5×16.5 mm, 6.5×17 mm,6.5×17.5 mm, 6.5×18 mm, 6.5×18.5 mm, 6.5×19 mm, 6.5×19.5 mm, or 6.5×20mm chambered areas.

The array may comprise at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90μm, 95 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, or500 μm spots. In some instances, the array comprises 70 μm spots.

The array may comprise at least about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm,45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, or 500 μm,525 μm, 550 μm, 575 μm, 600 μm, 625 μm, 650 μm, 675 μm, 700 μm, 725 μm,750 μm, 775 μm, 800 μm, 825 μm, 850 μm, 875 μm, 900 μm, 925 μm, 950 μm,975 μm, 1000 μm feature pitch. In some instances, the array comprises161 μm feature pitch.

The array may comprise one or more probes. In some instances, the arraycomprises at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,or 100 probes. Alternatively, the array comprises at least about 200,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, or 3000 probes. The array may comprise at least about 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,or 10000 probes. In some instances, the array comprises at least about960 probes. Alternatively, the array comprises at least about 2780probes. The probes may be specific for the plurality of oligonucleotidetags. The probes may be specific for at least a portion of the pluralityof oligonucleotide tags. The probes may be specific for at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or 100% of thetotal number of the plurality of oligonucleotide tags. Alternatively,the probes are specific for at least about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97% or 100% of the total number of differentoligonucleotide tags of the plurality of oligonucleotide tags. Theprobes may be oligonucleotides. The oligonucleotides may be at leastabout 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides long.In other instances, the probes are non-specific probes. For example, theprobes may be specific for a detectable label that is attached to thelabeled-molecule. The probe may be streptavidin.

The array may be a printed array. In some instances, the printed arraycomprises one or more oligonucleotides attached to a substrate. Forexample, the printed array comprises 5′ amine modified oligonucleotidesattached to an epoxy silane substrate.

Alternatively, the array comprises a slide with one or more wells. Theslide may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 wells. Alternatively, the slide comprises atleast about 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 650, 700,750, 800, 850, 900, 950, or 1000 wells. In some instances, the slidecomprises 16 wells. Alternatively, the slide comprises 96 wells. Inother instances, the slide comprises at least about 80, 160, 240, 320,400, 480, 560, 640, 720, 800, 880, or 960 wells.

In some instances, the solid support is an Affymetrix 3K tag array,Arrayjet non-contact printed array, or Applied Microarrays Inc (AMI)array. Alternatively, the support comprises a contact printer, impactprinter, dot printer, or pin printer.

The solid support may comprise the use of beads that self-assemble inmicrowells. For example, the solid support comprises Illumina'sBeadArray Technology. Alternatively, the solid support comprises AbbottMolecular's Bead Array technology, and Applied Microarray's FlexiPlex™system.

In other instances, the solid support is a plate. Examples of platesinclude, but are not limited to, MSD multi-array plates, MSD Multi-Spot®plates, microplate, ProteOn microplate, AlphaPlate, DELFIA plate,IsoPlate, and LumaPlate.

The method may further comprise attaching at least one of a plurality oflabeled nucleic acids to a support. The support may comprise a pluralityof beads. The support may comprise an array. The support may comprise aglass slide.

The glass slide may comprise one or more wells. The one or more wellsmay be etched on the glass slide. The one or more wells may comprise atleast 960 wells. The glass slide may comprise one or more probes. Theone or more probes may be printed onto the glass slide. The one or morewells may further comprise one or more probes. The one or more probesmay be printed within the one or more wells. The one or more probes maycomprise 960 nucleic acids.

The methods and kits disclosed herein may further comprise distributingthe plurality of first sample tags, the plurality of second sample tags,the plurality of molecular identifier labels, or any combination thereofin a microwell plate. The methods and kits disclosed herein may furthercomprise distributing one or more beads in the microwell plate. Themethods and kits disclosed herein may further comprise distributing theplurality of samples in a plurality of wells of a microwell plate. Theone or more of the plurality of samples may comprise a plurality ofcells. One or more of the plurality of samples may comprise a pluralityof nucleic acids. The method may further comprise distributing one orfewer cells to the plurality of wells. The plurality of cells may belysed in the microwell plate. The method may further comprisesynthesizing cDNA in the microwell plate. Synthesizing cDNA may comprisereverse transcription of mRNA. The microwell plate may comprise amicrowell plate fabricated on PDMS by soft lithography, etched on asilicon wafer, etched on a glass slide, patterned photoresist on a glassslide, or a combination thereof. The microwell may comprise a hole on amicrocapillary plate. The microwell plate may comprise a water-in-oilemulsion. The microwell plate may comprise at least one or more wells.The microwell plate may comprise at least about 6 wells, 12 wells, 48wells, 96 wells, 384 wells, 960 wells or 1000 wells.

The methods and kits may further comprise a chip. The microwell platemay be attached to the chip. The chip may comprise at least about 6wells, 12 wells, 48 wells, 96 wells, 384 wells, 960 wells, 1000 wells,2000 wells, 3000 wells, 4000 wells, 5000 wells, 6000 wells, 7000 wells,8000 wells, 9000 wells, 10,000 wells, 20,000 wells, 30,000 wells, 40,000wells, 50,000 wells, 60,000 wells, 70,000 wells, 80,000 wells, 90,000wells, 100,000 wells, 200,000 wells, 500,000 wells, or a million wells.The wells may comprise an area of at least about 300 microns², 400microns², 500 microns², 600 microns², 700 microns², 800 microns², 900microns², 1000 microns², 1100 microns², 1200 microns², 1300 microns²,1400 microns², 1500 microns². The method may further comprisedistributing between about 10,000 and 30,000 samples on the chip.

Functionalized Surfaces and Oligonucleotides

The bead may comprise a functionalized surface. A functionalized surfacemay refer to the surface of the solid support comprising a functionalgroup. A functional group may be a group capable of forming anattachment with another functional group. For example, a functionalgroup may be biotin, which may form an attachment with streptavidin,another functional group. Exemplary functional groups may include, butare not limited to, aldehydes, ketones, carboxy groups, amino groups,biotin, streptavidin, nucleic acids, small molecules (e.g., for clickchemistry), homo- and hetero-bifunctional reagents (e.g.,N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), dimaleimide,dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate(SATA), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl 4-(N-mafeimidomethyl)-cyclohexane-1-carboxylate (SMCC) and6-hydrazinonicotimide (HYNIC), and antibodies. In some instances thefunctional group is a carboxy group (e.g., COOH).

Oligonucleotides (e.g., nucleic acids) may be attached to functionalizedsolid supports. The immobilized oligonucleotides on solid supports orsimilar structures may serve as nucleic acid probes, and hybridizationassays may be conducted wherein specific target nucleic acids may bedetected in complex biological samples.

The solid support (e.g., beads) may be functionalized for theimmobilization of oligonucleotides. An oligonucleotide may be conjugatedto a solid support through a covalent amide bond formed between thesolid support and the oligonucleotide.

A support may be conjugated to at least about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or moreoligonucleotides. A support may be conjugated to at least about 100000,200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000or 10000000, 100000000, 500000000, 1000000000 or more oligonucleotides.A support may be conjugated to at least about 100000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000,3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000 or10000000, 100000000, 500000000, 1000000000 or more oligonucleotides. Asupport may be conjugated to at least 1 million oligonucleotides. Asupport may be conjugated to at least 10 million oligonucleotides. Asupport may be conjugated to at least 25 million oligonucleotides. Asupport may be conjugated to at least 50 million oligonucleotides. Asupport may be conjugated to at least 100 million oligonucleotides. Asupport may be conjugated to at least 250 million oligonucleotides. Asupport may be conjugated to at least 500 million oligonucleotides. Asupport may be conjugated to at least 750 million oligonucleotides. Asupport may be conjugated to at least about 1, 2, 3 4, 5, 6, 7, 8, 9,10, 11, 12 13, 14, or 15 billion oligonucleotides. A support may beconjugated to at least 1 billion oligonucleotides. A support may beconjugated to at least 5 billion oligonucleotides.

The oligonucleotides may be attached to the support (e.g., beads,polymers, gels) via a linker. Conjugation may comprise covalent ornon-covalent attachment. Conjugation may introduce a variable spacerbetween the beads and the nucleic acids. The linker between the supportand the oligonucleotide may be cleavable (e.g., photocleavable linkage,acid labile linker, heat sensitive linker, and enzymatically cleavablelinker).

Cross-linking agents for use for conjugating molecules to supports mayinclude agents capable of reacting with a functional group present on asurface of the solid support and with a functional group present in themolecule. Reagents capable of such reactivity may include aldehydes,ketones, carboxy groups, amino groups, biotin, streptavidin, nucleicacids, small molecules (e.g., for click chemistry), homo- andhetero-bifunctional reagents (e.g., N-succinimidyl(4-iodoacetyl)aminobenzoate (STAB), dimaleimide, dithio-bis-nitrobenzoic acid (DTNB),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl4-(N-mafeimidomethyl)-cyclohexane-1-carboxylate (SMCC) and6-hydrazinonicotimide (HYNIC).

A bead may be functionalized with a carboxy functional group and anoligonucleotide may be functionalized with an amino functional group.

A support may be smooth. Alternatively, or additionally, a support maycomprise divots, ridges, or wells. A support may comprise a microwellarray. A microwell array may be functionalized with functional groupsthat facilitate the attachment of oligonucleotides. The functionalgroups on the microwell array may be different for different positionson the microwell array. The functional groups on the microwell array maybe the same for all regions of the microwell array.

Assay System Components

Microwell Arrays

As described above, microwell arrays are used to entrap single cells andbeads (one bead per cell) within a small reaction chamber of definedvolume. Each bead comprises a library of oligonucleotide probes for usein stochastic labeling and digital counting of the entire complement ofcellular mRNA molecules, which are released upon lysis of the cell. Inone embodiment of the present disclosure, the microwell arrays are aconsumable component of the assay system. In other embodiments, themicrowell arrays may be reusable. In either case, they may be configuredto be used as a stand-alone device for use in performing assaysmanually, or they may be configured to comprise a removable or fixedcomponent of an instrument that provides for full or partial automationof the assay procedure.

The microwells of the array can be fabricated in a variety of shapes andsizes, which are chosen to optimize the efficiency of trapping a singlecell and bead in each well. Appropriate well geometries include, but arenot limited to, cylindrical, conical, hemispherical, rectangular, orpolyhedral (e.g., three dimensional geometries comprised of severalplanar faces, for example, hexagonal columns, octagonal columns,inverted triangular pyramids, inverted square pyramids, invertedpentagonal pyramids, inverted hexagonal pyramids, or inverted truncatedpyramids). The microwells may comprise a shape that combines two or moreof these geometries. For example, in one embodiment it may be partlycylindrical, with the remainder having the shape of an inverted cone. Inanother embodiment, it may include two side-by-side cylinders, one oflarger diameter than the other, that are connected by a vertical channel(that is, parallel to the cylinder axes) that extends the full length(depth) of the cylinders. In general, the open end (or mouth) of eachmicrowell will be located at an upper surface of the microwell array,but in some embodiments the openings may be located at a lower surfaceof the array. In general, the closed end (or bottom) of the microwellwill be flat, but curved surfaces (e.g., convex or concave) are alsopossible. In general, the shape (and size) of the microwells will bedetermined based on the types of cells and/or beads to be trapped in themicrowells.

Microwell dimensions may be characterized in terms of the diameter anddepth of the well. As used herein, the diameter of the microwell refersto the largest circle that can be inscribed within the planarcross-section of the microwell geometry. In one embodiment of thepresent disclosure, the diameter of the microwells may range from about0.1 to about 5-fold the diameter of the cells and/or beads to be trappedwithin the microwells. In other embodiments, the microwell diameter isat least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 2-fold,at least 3-fold, at least 4-fold, or at least 5-fold the diameter of thecells and/or beads to be trapped within the microwells. In yet otherembodiments, the microwell diameter is at most 5-fold, at most 4-fold,at most 3-fold, at most 2-fold, at most 1-fold, at most 0.5-fold, or atmost 0.1-fold the diameter of the cells and/or beads to be trappedwithin the microwells. In one embodiment, the microwell diameter isabout 2.5-fold the diameter of the cells and/or beads to be trappedwithin the microwells. Those of skill in the art will appreciate thatthe microwell diameter may fall within any range bounded by any of thesevalues (e.g., from about 0.2-fold to about 3.5-fold the diameter of thecells and/or beads to be trapped within the microwells). Alternatively,the diameter of the microwells can be specified in terms of absolutedimensions. In one embodiment of the present disclosure, the diameter ofthe microwells may range from about 5 to about 50 microns. In otherembodiments, the microwell diameter is at least 5 microns, at least 10microns, at least 15 microns, at least 20 microns, at least 25 microns,at least 30 microns, at least 35 microns, at least 40 microns, at least45 microns, or at least 50 microns. In yet other embodiments, themicrowell diameter is at most 50 microns, at most 45 microns, at most 40microns, at most 35 microns, at most 30 microns, at most 25 microns, atmost 20 microns, at most 15 microns, at most 10 microns, or at most 5microns. In one embodiment, the microwell diameter is about 30 microns.Those of skill in the art will appreciate that the microwell diametermay fall within any range bounded by any of these values (e.g., fromabout 28 microns to about 34 microns).

The microwell depth is chosen to optimize cell and bead trappingefficiency while also providing efficient exchange of assay buffers andother reagents contained within the wells. In one embodiment of thepresent disclosure, the depth of the microwells may range from about 0.1to about 5-fold the diameter of the cells and/or beads to be trappedwithin the microwells. In other embodiments, the microwell depth is atleast 0.1-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, atleast 3-fold, at least 4-fold, or at least 5-fold the diameter of thecells and/or beads to be trapped within the microwells. In yet otherembodiments, the microwell depth is at most 5-fold, at most 4-fold, atmost 3-fold, at most 2-fold, at most 1-fold, at most 0.5-fold, or atmost 0.1-fold the diameter of the cells and/or beads to be trappedwithin the microwells. In one embodiment, the microwell depth is about2.5-fold the diameter of the cells and/or beads to be trapped within themicrowells. Those of skill in the art will appreciate that the microwelldepth may fall within any range bounded by any of these values (e.g.,from about 0.2-fold to about 3.5-fold the diameter of the cells and/orbeads to be trapped within the microwells). Alternatively, the diameterof the microwells can be specified in terms of absolute dimensions. Inone embodiment of the present disclosure, the depth of the microwellsmay range from about 10 to about 60 microns. In other embodiments, themicrowell depth is at least 10 microns, at least 20 microns, at least 25microns, at least 30 microns, at least 35 microns, at least 40 microns,at least 50 microns, or at least 60 microns. In yet other embodiments,the microwell depth is at most 60 microns, at most 50 microns, at most40 microns, at most 35 microns, at most 30 microns, at most 25 microns,at most 20 microns, or at most 10 microns. In one embodiment, themicrowell depth is about 30 microns. Those of skill in the art willappreciate that the microwell depth may fall within any range bounded byany of these values (e.g., from about 24 microns to about 36 microns).

The wells of the microwell array are arranged in a one dimensional, twodimensional, or three dimensional array, where three dimensional arraysmay be achieved, for example, by stacking a series of two or more twodimensional arrays (that is, by stacking two or more substratescomprising microwell arrays). The pattern and spacing between wells ischosen to optimize the efficiency of trapping a single cell and bead ineach well, as well as to maximize the number of wells per unit area ofthe array. The wells may be distributed according to a variety of randomor non-random patterns, for example, they may be distributed entirelyrandomly across the surface of the array substrate, or they may bearranged in a square grid, rectangular grid, or hexagonal grid. In oneembodiment of the present disclosure, the center-to-center distance (orspacing) between wells may vary from about 15 microns to about 75microns. In other embodiments, the spacing between wells is at least 15microns, at least 20 microns, at least 25 microns, at least 30 microns,at least 35 microns, at least 40 microns, at least 45 microns, at least50 microns, at least 55 microns, at least 60 microns, at least 65microns, at least 70 microns, or at least 75 microns. In yet otherembodiments, the microwell spacing is at most 75 microns, at most 70microns, at most 65 microns, at most 60 microns, at most 55 microns, atmost 50 microns, at most 45 microns, at most 40 microns, at most 35microns, at most 30 microns, at most 25 microns, at most 20 microns, orat most 15 microns. In one embodiment, the microwell spacing is about 55microns. Those of skill in the art will appreciate that the microwelldepth may fall within any range bounded by any of these values (e.g.,from about 18 microns to about 72 microns).

The microwell array may comprise surface features between the microwellsthat are designed to help guide cells and beads into the wells and/orprevent them from settling on the surfaces between wells. Examples ofsuitable surface features include, but are not limited to, domed,ridged, or peaked surface features that encircle the wells and/orstraddle the surface between wells.

The total number of wells in the microwell array is determined by thepattern and spacing of the wells and the overall dimensions of thearray. In one embodiment of the present disclosure, the number ofmicrowells in the array may range from about 96 to about 5,000,000 ormore. In other embodiments, the number of microwells in the array is atleast 96, at least 384, at least 1,536, at least 5,000, at least 10,000,at least 25,000, at least 50,000, at least 75,000, at least 100,000, atleast 500,000, at least 1,000,000, or at least 5,000,000. In yet otherembodiments, the number of microwells in the array is at most 5,000,000,at most 1,000,000, at most 75,000, at most 50,000, at most 25,000, atmost 10,000, at most 5,000, at most 1,536, at most 384, or at most 96wells. In one embodiment, the number of microwells in the array is about96. In another embodiment, the number of microwells is about 150,000.Those of skill in the art will appreciate that the number of microwellsin the array may fall within any range bounded by any of these values(e.g., from about 100 to 325,000).

Microwell arrays may be fabricated using any of a number of fabricationtechniques known to those of skill in the art. Examples of fabricationmethods that may be used include, but are not limited to, bulkmicromachining techniques such as photolithography and wet chemicaletching, plasma etching, or deep reactive ion etching; micro-molding andmicro-embossing; laser micromachining; 3D printing or other direct writefabrication processes using curable materials; and similar techniques.

Microwell arrays may be fabricated from any of a number of substratematerials known to those of skill in the art, where the choice ofmaterial typically depends on the choice of fabrication technique, andvice versa. Examples of suitable materials include, but are not limitedto, silicon, fused-silica, glass, polymers (e.g., agarose, gelatin,hydrogels, polydimethylsiloxane (PDMS; elastomer),polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP),polyethylene (PE), high density polyethylene (HDPE), polyimide, cyclicolefin polymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET), and epoxy resins), metals or metal films (e.g.,aluminum, stainless steel, copper, nickel, chromium, and titanium), andthe like. Typically, a hydrophilic material is desirable for fabricationof the microwell arrays (to enhance wettability and minimizenon-specific binding of cells and other biological material), buthydrophobic materials that can be treated or coated (e.g., by oxygenplasma treatment, or grafting of a polyethylene oxide surface layer) canalso be used. The use of porous, hydrophilic materials for thefabrication of the microwell array may be desirable in order tofacilitate capillary wicking/venting of entrapped air bubbles in thedevice. In some embodiments, the microwell array is fabricated with anoptical adhesive. In some embodiments, the microwell array is fabricatedwith a plasma or corona treated material. The use of plasma or coronatreated materials can make the material hydrophillic. In someembodiments, plasma or corona treated materials, such as a hydrophillicmaterial, can be more stable than non-treated materials. In someembodiments, the microwell array is fabricated from a single material.In other embodiments, the microwell array may comprise two or moredifferent materials that have been bonded together or mechanicallyjoined.

A variety of surface treatments and surface modification techniques maybe used to alter the properties of microwell array surfaces. Examplesinclude, but are not limited to, oxygen plasma treatments to renderhydrophobic material surfaces more hydrophilic, the use of wet or dryetching techniques to smooth (or roughen) glass and silicon surfaces,adsorption and/or grafting of polyethylene oxide or other polymer layersto substrate surfaces to render them more hydrophilic and less prone tonon-specific adsorption of biomolecules and cells, the use of silanereactions to graft chemically-reactive functional groups to otherwiseinert silicon and glass surfaces, etc. Photodeprotection techniques canbe used to selectively activate chemically-reactive functional groups atspecific locations in the array structure, for example, the selectiveaddition or activation of chemically-reactive functional groups such asprimary amines or carboxyl groups on the inner walls of the microwellsmay be used to covalently couple oligonucleotide probes, peptides,proteins, or other biomolecules to the walls of the microwells. Ingeneral, the choice of surface treatment or surface modificationutilized will depend both on the type of surface property that isdesired and on the type of material from which the microwell array ismade.

In some embodiments, it may be advantageous to seal the openings ofmicrowells during, for example, cell lysis steps, to prevent crosshybridization of target nucleic acid between adjacent microwells. Amicrowell may be sealed using a cap such as a solid support or a bead,where the diameter of the bead is larger than the diameter of themicrowell. For example, a bead used as a cap can be at least about 10,20, 30, 40, 50, 60, 70, 80 or 90% larger than the diameter of themicrowell. Alternatively, a cap may be at most about 10, 20, 30, 40, 50,60, 70, 80 or 90% larger than the diameter of the microwell.

A bead used as a cap may comprise cross-linked dextran beads (e.g.,Sephadex). Cross-linked dextran can range from about 10 micrometers toabout 80 micrometers. The cross-linked dextran of the bead cap can befrom 20 micrometers to about 50 micrometers. A cap can comprise, forexample, inorganic nanopore membranes (e.g., aluminum oxides), dialysismembranes, glass slides, coverslips, and/or hydrophilic plastic film(e.g., film coated with a thin film of agarose hydrated with lysisbuffer).

In some embodiments, the cap may allow buffer to pass into and out ofthe microwell, while preventing macromolecules (e.g., nucleic acids)from migrating out of the well. A macromolecule of at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or morenucleotides can be blocked from migrating into or out of the microwellby the cap. A macromolecule of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides can beblocked from migrating into or out of the microwell by the cap.

In some embodiments, a sealed microwell array can comprise a singlelayer of beads on top of the microwells. In some embodiments, a sealedmicrowell array can comprise multiple layers of beads on top of themicrowells. A sealed microwell array can comprise about 1, 2, 3, 4, 5,or 6 or more layers of beads.

Mechanical Fixtures

When performing multiplexed, single cell stochastic labeling/molecularindexing assays manually, it is convenient to mount the microwell arrayin a mechanical fixture to create a reaction chamber and facilitate thepipetting or dispensing of cell suspensions and assay reagents onto thearray (FIGS. 69 and 70). In the example illustrated in FIG. 69, thefixture accepts a microwell array fabricated on a 1 mm thick substrate,and provides mechanical support in the form of a silicone gasket toconfine the assay reagents to a reaction chamber that is 16 mm wide×35mm long×approximately 4 mm deep, thereby enabling the use of 800microliters to 1 milliliter of cell suspension and bead suspension(comprising bead-based oligonucleotide labels) to perform the assay.

The fixture consists of rigid, machined top and bottom plates (e.g.,aluminum) and a compressible (e.g., silicone, polydimethylsiloxane)gasket for creating the walls of the chamber or well. Design featuresinclude: (i) Chamfered aperture edges and clearance for rotatingmicroscope objectives in and out of position as needed (for viewing themicrowell array at different magnifications). (ii) Controlledcompression of the silicone gasket to ensure uniform, repeatableformation of a leak-proof seal with the microwell array substrate. (iii)Captive fasteners for convenient operation. (iv) A locating clampmechanism for secure and repeatable positioning of the array. (v)Convenient disassembly for removal of the array during rinse steps.

The top and bottom plates may be fabricated using any of a variety oftechniques (e.g., conventional machining, CNC machining, injectionmolding, 3D printing, etc.) using a variety of materials (e.g.,aluminum, anodized aluminum, stainless steel, teflon,polymethylmethacrylate (PMMA), polycarbonate (PC), or similar rigidpolymer materials).

The silicone (polydimethylsiloxane; PDMS) gasket may be configured tocreate multiple chambers (see FIG. 71) in order to run controls andexperiments (or replicate experiments, or multiple independentexperiments) in parallel. The gasket is molded from PDMS or similarelastomeric material using a Teflon mold that includes draft angles forthe vertical gasket walls to provide for good release characteristics.Alternatively, molds can be machined from aluminum or other materials(e.g., black delrin, polyetherimide (ultem), etc.), and coated withTeflon if necessary to provide for good release characteristics. Thegasket mold designs are inverted, i.e. so that the top surface of themolded part (i.e. the surface at the interface with a glass slide orsilicon wafer used to cover the mold during casting) becomes the surfacefor creating a seal with the microwell array substrate during use,thereby avoiding potential problems with mold surface roughness andsurface contamination in creating a smooth gasket surface (to ensure aleak-proof seal with the array substrate), and also providing for aflexible choice of substrate materials and the option of pre-assembly byusing the microwell array substrate as a base during casting. The gasketmold designs may also include force focusing ridges at the boundaries ofthe well areas, i.e. the central mesa(s) in the mold (which form thewell(s)) have raised ridges at the locations which become the perimeterof the well(s), so that a cover placed on top of the mold after fillingrests on a small contact area at the precise location where good edgeprofile is critical for forming a leak-proof seal between the gasket andsubstrate during use.

Instrument Systems

The present disclosure also includes instrument systems and consumablesto support the automation of multiplexed, single cell stochasticlabeling/molecular indexing assays. Such systems may include consumablecartridges that incorporate microwell arrays integrated with flow cells,as well as the instrumentation necessary to provide control and analysisfunctionality such as (i) fluidics control, (ii) temperature control,(iii) cell and/or bead distribution and collection mechanisms, (iv) celllysis mechanisms, (v) imaging capability, and (vi) image processing. Insome embodiments, the input for the system comprises a cell sample andthe output comprises a bead suspension comprising beads having attachedoligonucleotides that incorporate sample tags, cell tags, and molecularindexing tags. In other embodiments, the system may include additionalfunctionality, such as thermal cycling capability for performing PCRamplification, in which case the input for the system comprises a cellsample and the output comprises an oligonucleotide library resultingfrom amplification of the oligonucleotides incorporating sample tags,cell tags, and molecular indexing tags that were originally attached tobeads. In yet other embodiments, the system may also include sequencingcapability, with or without the need for oligonucleotide amplification,in which case the input for the system is a cell sample and the outputcomprises a dataset further comprising the sequences of all sample tag,cell tag, and molecular indexing tags associated with the targetsequences of interest.

Microwell Array Flow Cells

In many embodiments of the automated assay system, the microwell arraysubstrate will be packaged within a flow cell that provides forconvenient interfacing with the rest of the fluid handling system andfacilitates the exchange of fluids, e.g., cell and bead suspensions,lysis buffers, rinse buffers, etc., that are delivered to the microwellarray. Design features may include: (i) one or more inlet ports forintroducing cell samples, bead suspensions, and/or other assay reagents,(ii) one or more microwell array chambers designed to provide foruniform filling and efficient fluid-exchange while minimizing backeddies or dead zones, and (iii) one or more outlet ports for delivery offluids to a sample collection point and/or a waste reservoir. In someembodiments, the design of the flow cell may include a plurality ofmicroarray chambers that interface with a plurality of microwell arrayssuch that one or more cell samples may be processed in parallel. In someembodiments, the design of the flow cell may further include featuresfor creating uniform flow velocity profiles, i.e. “plug flow”, acrossthe width of the array chamber to provide for more uniform delivery ofcells and beads to the microwells, for example, by using a porousbarrier located near the chamber inlet and upstream of the microwellarray as a “flow diffuser”, or by dividing each array chamber intoseveral subsections that collectively cover the same total array area,but through which the divided inlet fluid stream flows in parallel. Insome embodiments, the flow cell may enclose or incorporate more than onemicrowell array substrate. In some embodiments, the integrated microwellarray/flow cell assembly may constitute a fixed component of the system.In some embodiments, the microwell array/flow cell assembly may beremovable from the instrument.

In general, the dimensions of fluid channels and the array chamber(s) inflow cell designs will be optimized to (i) provide uniform delivery ofcells and beads to the microwell array, and (ii) to minimize sample andreagent consumption. In some embodiments, the width of fluid channelswill be between 50 microns and 20 mm. In other embodiments, the width offluid channels may be at least 50 microns, at least 100 microns, atleast 200 microns, at least 300 microns, at least 400 microns, at least500 microns, at least 750 microns, at least 1 mm, at least 2.5 mm, atleast 5 mm, at least 10 mm, or at least 20 mm. In yet other embodiments,the width of fluid channels may at most 20 mm, at most 10 mm, at most 5mm, at most 2.5 mm, at most 1 mm, at most 750 microns, at most 500microns, at most 400 microns, at most 300 microns, at most 200 microns,at most 100 microns, or at most 50 microns. In one embodiment, the widthof fluid channels is about 2 mm. Those of skill in the art willappreciate that the width of the fluid channels may fall within anyrange bounded by any of these values (e.g., from about 250 microns toabout 3 mm).

In some embodiments, the depth of the fluid channels will be between 50microns and 10 mm. In other embodiments, the depth of fluid channels maybe at least 50 microns, at least 100 microns, at least 200 microns, atleast 300 microns, at least 400 microns, at least 500 microns, at least750 microns, at least 1 mm, at least 1.25 mm, at least 1.5 mm, at least1.75 mm, at least 2 mm, at least 2.5 mm, at least 3 mm, at least 3.5 mm,at least 4 mm, at least 4.5 mm, at least 5 mm, at least 5.5 mm, at least6 mm, at least 6.5 mm, at least 7 mm, at least 7.5 mm, at least 8 mm, atleast 8.5 mm, at least 9 mm, or at least 9.5 mm. In other embodiments,the depth of fluid channels may be at most 10 mm, at most 9.5 mm, atmost 9 mm, at most 8.5 mm, at most 8 mm, at most 7.5 mm, at most 7 mm,at most 6.5 mm, at most 6 mm, at most 5.5 mm, at most 5 mm, at most 4.5mm, at most 4 mm, at most 3.5 mm, at most 3 mm, at most 2 mm, at most1.75 mm, at most 1.5 mm, at most 1.25 mm, at most 1 mm, at most 750microns, at most 500 microns, at most 400 microns, at most 300 microns,at most 200 microns, at most 100 microns, or at most 50 microns. In oneembodiment, the depth of the fluid channels is about 1 mm. Those ofskill in the art will appreciate that the depth of the fluid channelsmay fall within any range bounded by any of these values (e.g., fromabout 800 microns to about 1 mm).

Flow cells may be fabricated using a variety of techniques and materialsknown to those of skill in the art. In general, the flow cell will befabricated as a separate part and subsequently either mechanicallyclamped or permanently bonded to the microwell array substrate. Examplesof suitable fabrication techniques include conventional machining, CNCmachining, injection molding, 3D printing, alignment and lamination ofone or more layers of laser or die-cut polymer films, or any of a numberof microfabrication techniques such as photolithography and wet chemicaletching, dry etching, deep reactive ion etching, or laser micromachiningOnce the flow cell part has been fabricated it may be attached to themicrowell array substrate mechanically, e.g., by clamping it against themicrowell array substrate (with or without the use of a gasket), or itmay be bonded directly to the microwell array substrate using any of avariety of techniques (depending on the choice of materials used) knownto those of skill in the art, for example, through the use of anodicbonding, thermal bonding, ultrasonic welding, or any of a variety ofadhesives or adhesive films, including epoxy-based, acrylic-based,silicone-based, UV curable, polyurethane-based, or cyanoacrylate-basedadhesives.

Flow cells may be fabricated using a variety of materials known to thoseof skill in the art. Examples of suitable materials include, but are notlimited to, silicon, fused-silica, glass, any of a variety of polymers,e.g., polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate(PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), highdensity polyethylene (HDPE), polyimide, cyclic olefin polymers (COP),cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxyresins, metals (e.g., aluminum, stainless steel, copper, nickel,chromium, and titanium), or a combination of these materials.

Cartridges

In many embodiments of the automated assay system, the microwell array,with or without an attached flow cell, will be packaged within aconsumable cartridge that interfaces with the instrument system andwhich may incorporate additional functionality. Design features ofcartridges may include (i) one or more inlet ports for creating fluidconnections with the instrument and/or manually introducing cellsamples, bead suspensions, and/or other assay reagents into thecartridge, (ii) one or more bypass channels, i.e. for self-metering ofcell samples and bead suspensions, to avoid overfilling and/or backflow, (iii) one or more integrated microwell array/flow cell assemblies,or one or more chambers within which the microarray substrate(s) arepositioned, (iv) integrated miniature pumps or other fluid actuationmechanisms for controlling fluid flow through the device, (v) integratedminiature valves for compartmentalizing pre-loaded reagents and/orcontrolling fluid flow through the device, (vi) vents for providing anescape path for trapped air, (vii) one or more sample and reagent wastereservoirs, (viii) one or more outlet ports for creating fluidconnections with the instrument and/or providing a processed samplecollection point, (ix) mechanical interface features for reproduciblypositioning the removable, consumable cartridge with respect to theinstrument system, and for providing access so that external magnets canbe brought into close proximity with the microwell array, (x) integratedtemperature control components and/or a thermal interface for providinggood thermal contact with the instrument system, and (xi) opticalinterface features, e.g., a transparent window, for use in opticalinterrogation of the microwell array. In some embodiments, the cartridgeis designed to process more than one sample in parallel. In someembodiments of the device, the cartridge may further comprise one ormore removable sample collection chamber(s) that are suitable forinterfacing with stand-alone PCR thermal cyclers and/or sequencinginstruments. In some embodiments of the device, the cartridge itself issuitable for interfacing with stand-alone PCR thermal cyclers and/orsequencing instruments.

In some embodiments of the device, the cartridge may further comprisecomponents that are designed to create physical and/or chemical barriersthat prevent diffusion of (or increase path lengths and diffusion timesfor) large molecules in order to minimize cross-contamination betweenmicrowells. Examples of such barriers include, but are not limited to, apattern of serpentine channels used for delivery of cells and beads tothe microwell array, a retractable platen or deformable membrane that ispressed into contact with the surface of the microwell array substrateduring lysis or incubation steps, the use of larger beads, e.g.,Sephadex beads as described previously, to block the openings of themicrowells, or the release of an immiscible, hydrophobic fluid from areservoir within the cartridge during lysis or incubation steps, toeffectively separate and compartmentalize each microwell in the array.Any or all of these barriers, or an embodiment without such barriers,may be combined with raising the viscosity of the solution in andadjacent to the microwells, e.g., through the addition of solutioncomponents such as glycerol or polyethylene glycol.

In general, the dimensions of fluid channels and the array chamber(s) incartridge designs will be optimized to (i) provide uniform delivery ofcells and beads to the microwell array, and (ii) to minimize sample andreagent consumption. In some embodiments, the width of fluid channelswill be between 50 microns and 20 mm. In other embodiments, the width offluid channels may be at least 50 microns, at least 100 microns, atleast 200 microns, at least 300 microns, at least 400 microns, at least500 microns, at least 750 microns, at least 1 mm, at least 2.5 mm, atleast 5 mm, at least 10 mm, or at least 20 mm. In yet other embodiments,the width of fluid channels may at most 20 mm, at most 10 mm, at most 5mm, at most 2.5 mm, at most 1 mm, at most 750 microns, at most 500microns, at most 400 microns, at most 300 microns, at most 200 microns,at most 100 microns, or at most 50 microns. In one embodiment, the widthof fluid channels is about 2 mm. Those of skill in the art willappreciate that the width of the fluid channels may fall within anyrange bounded by any of these values (e.g., from about 250 microns toabout 3 mm).

In some embodiments, the depth of the fluid channels in cartridgedesigns will be between 50 microns and 10 mm. In other embodiments, thedepth of fluid channels may be at least 50 microns, at least 100microns, at least 200 microns, at least 300 microns, at least 400microns, at least 500 microns, at least 750 microns, at least 1 mm, atleast 1.25 mm, at least 1.5 mm, at least 1.75 mm, at least 2 mm, atleast 2.5 mm, at least 3 mm, at least 3.5 mm, at least 4 mm, at least4.5 mm, at least 5 mm, at least 5.5 mm, at least 6 mm, at least 6.5 mm,at least 7 mm, at least 7.5 mm, at least 8 mm, at least 8.5 mm, at least9 mm, or at least 9.5 mm. In yet other embodiments, the depth of fluidchannels may be at most 10 mm, at most 9.5 mm, at most 9 mm, at most 8.5mm, at most 8 mm, at most 7.5 mm, at most 7 mm, at most 6.5 mm, at most6 mm, at most 5.5 mm, at most 5 mm, at most 4.5 mm, at most 4 mm, atmost 3.5 mm, at most 3 mm, at most 2 mm, at most 1.75 mm, at most 1.5mm, at most 1.25 mm, at most 1 mm, at most 750 microns, at most 500microns, at most 400 microns, at most 300 microns, at most 200 microns,at most 100 microns, or at most 50 microns. In one embodiment, the depthof the fluid channels is about 1 mm. Those of skill in the art willappreciate that the depth of the fluid channels may fall within anyrange bounded by any of these values (e.g., from about 800 microns toabout 1 mm).

Cartridges may be fabricated using a variety of techniques and materialsknown to those of skill in the art. In general, the cartridges will befabricated as a series of separate component parts (FIG. 72) andsubsequently assembled (FIGS. 72 and 73) using any of a number ofmechanical assembly or bonding techniques. Examples of suitablefabrication techniques include, but are not limited to, conventionalmachining, CNC machining, injection molding, thermoforming, and 3Dprinting. Once the cartridge components have been fabricated they may bemechanically assembled using screws, clips, and the like, or permanentlybonded using any of a variety of techniques (depending on the choice ofmaterials used), for example, through the use of thermal or ultrasonicbonding/welding or any of a variety of adhesives or adhesive films,including epoxy-based, acrylic-based, silicone-based, UV curable,polyurethane-based, or cyanoacrylate-based adhesives.

Cartridge components may be fabricated using any of a number of suitablematerials, including but not limited to silicon, fused-silica, glass,any of a variety of polymers, e.g., polydimethylsiloxane (PDMS;elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC),polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE),polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC),polyethylene terephthalate (PET), epoxy resins, or metals (e.g.,aluminum, stainless steel, copper, nickel, chromium, and titanium).

As described above, the inlet and outlet features of the cartridge maybe designed to provide convenient and leak-proof fluid connections withthe instrument, or may serve as open reservoirs for manual pipetting ofsamples and reagents into or out of the cartridge. Examples ofconvenient mechanical designs for the inlet and outlet port connectorsinclude, but are not limited to, threaded connectors, swaged connectors,Luer lock connectors, Luer slip or “slip tip” connectors, press fitconnectors, and the like. In some embodiments, the inlet and outletports of the cartridge may further comprise caps, spring-loaded coversor closures, phase change materials, or polymer membranes that may beopened or punctured when the cartridge is positioned in the instrument,and which serve to prevent contamination of internal cartridge surfacesduring storage and/or which prevent fluids from spilling when thecartridge is removed from the instrument. As indicated above, in someembodiments the one or more outlet ports of the cartridge may furthercomprise a removable sample collection chamber that is suitable forinterfacing with stand-alone PCR thermal cyclers and/or sequencinginstruments.

As indicated above, in some embodiments the cartridge may includeintegrated miniature pumps or other fluid actuation mechanisms forcontrol of fluid flow through the device. Examples of suitable miniaturepumps or fluid actuation mechanisms include, but are not limited to,electromechanically- or pneumatically-actuated miniature syringe orplunger mechanisms, chemical propellants, membrane diaphragm pumpsactuated pneumatically or by an external piston, pneumatically-actuatedreagent pouches or bladders, or electro-osmotic pumps.

As described above, in some embodiments the cartridge may includeminiature valves for compartmentalizing pre-loaded reagents and/orcontrolling fluid flow through the device. Examples of suitableminiature valves include, but are not limited to, one-shot “valves”fabricated using wax or polymer plugs that can be melted or dissolved,or polymer membranes that can be punctured; pinch valves constructedusing a deformable membrane and pneumatic, hydraulic, magnetic,electromagnetic, or electromechanical (solenoid) acutation, one-wayvalves constructed using deformable membrane flaps, and miniature gatevalves.

As indicated above, in some embodiments the cartridge may include ventsfor providing an escape path for trapped air. Vents may be constructedaccording to a variety of techniques known to those of skill in the art,for example, using a porous plug of polydimethylsiloxane (PDMS) or otherhydrophobic material that allows for capillary wicking of air but blockspenetration by water. Vents may also be constructed as apertures throughhydrophobic barrier materials, such that wetting to the aperture wallsdoes not occur at the pressures used during operation.

In general, the mechanical interface features of the cartridge providefor easily removable but highly precise and repeatable positioning ofthe cartridge relative to the instrument system. Suitable mechanicalinterface features include, but are not limited to, alignment pins,alignment guides, mechanical stops, and the like. In some embodiments,the mechanical design features will include relief features for bringingexternal apparatus, e.g., magnets or optical components, into closeproximity with the microwell array chamber (FIG. 72).

In some embodiments, the cartridge will also include temperature controlcomponents or thermal interface features for mating to externaltemperature control modules. Examples of suitable temperature controlelements include, but are not limited to, resistive heating elements,miniature infrared-emitting light sources, Peltier heating or coolingdevices, heat sinks, thermistors, thermocouples, and the like. Thermalinterface features will typically be fabricated from materials that aregood thermal conductors (e.g., copper, gold, silver, aluminium, etc.)and will typically comprise one or more flat surfaces capable of makinggood thermal contact with external heating blocks or cooling blocks.

In many embodiments, the cartridge will include optical interfacefeatures for use in optical imaging or spectroscopic interrogation ofthe microwell array. Typically, the cartridge will include an opticallytransparent window, e.g., the microwell substrate itself or the side ofthe flow cell or microarray chamber that is opposite the microwellarray, fabricated from a material that meets the spectral requirementsfor the imaging or spectroscopic technique used to probe the microwellarray. Examples of suitable optical window materials include, but arenot limited to, glass, fused-silica, polymethylmethacrylate (PMMA),polycarbonate (PC), cyclic olefin polymers (COP), or cyclic olefincopolymers (COC). Typically, the cartridge will include a secondoptically transparent or translucent window or region which can be usedto illuminate the microwell array in transverse, reflected, or obliqueillumination orientations.

Instruments

The present disclosure also includes instruments for use in theautomation of multiplexed, single cell stochastic labeling/molecularindexing assays. As indicated above, these instruments may providecontrol and analysis functionality such as (i) fluidics control, (ii)temperature control, (iii) cell and/or bead distribution and collectionmechanisms, (iv) cell lysis mechanisms, (v) magnetic field control, (vi)imaging capability, and (vii) image processing. In some embodiments, theinstrument system may comprise one or more modules (one possibleembodiment of which is illustrated schematically in FIG. 74), where eachmodule provides one or more specific functional feature sets to thesystem. In other embodiments, the instrument system may be packaged suchthat all system functionality resides within the same package. FIG. 75provides a schematic illustration of the process steps included in oneembodiment of the automated system. As indicated above, in someembodiments, the system may comprise additional functional units, eitheras integrated components or as modular components of the system, thatexpand the functional capabilities of the system to include PCRamplification (or other types of oligonucleotide amplificationtechniques) and oligonucleotide sequencing.

In general, the instrument system will provide fluidics capability fordelivering samples and/or reagents to the one or more microarraychamber(s) or flow cell(s) within one or more assay cartridge(s)connected to the system. Assay reagents and buffers may be stored inbottles, reagent and buffer cartridges, or other suitable containersthat are connected to the cartridge inlets. The system may also includewaste reservoirs in the form of bottles, waste cartridges, or othersuitable waste containers for collecting fluids downstream of the assaycartridge(s). Control of fluid flow through the system will typically beperformed through the use of pumps (or other fluid actuation mechanisms)and valves. Examples of suitable pumps include, but are not limited to,syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragmpumps, and the like. In some embodiments, fluid flow through the systemmay be controlled by means of applying positive pneumatic pressure atthe one or more inlets of the reagent and buffer containers, or at theinlets of the assay cartridge(s). In some embodiments, fluid flowthrough the system may be controlled by means of drawing a vacuum at theone or more outlets of the waste reservoirs, or at the outlets of theassay cartridge(s). Examples of suitable valves include, but are notlimited to, check valves, electromechanical two-way or three-way valves,pneumatic two-way and three-way valves, and the like. In someembodiments, pulsatile flow may be applied during assay wash/rinse stepsto facilitate complete and efficient exchange of fluids within the oneor more microwell array flow cell(s) or chamber(s).

As indicated above, in some embodiments the instrument system mayinclude mechanisms for further facilitating the uniform distribution ofcells and beads over the microwell array. Examples of such mechanismsinclude, but are not limited to, rocking, shaking, swirling,recirculating flow, low frequency agitation (for example, using a rockerplate or through pulsing of a flexible (e.g., silicone) membrane thatforms a wall of the chamber or nearby fluid channel), or high frequencyagitation (for example, through the use of piezoelectric transducers).In some embodiments, one or more of these mechanisms is utilized incombination with physical structures or features on the interior wallsof the flow cell or array chamber, e.g., mezzanine/top hat structures,chevrons, or ridge arrays, to facilitate mixing and/or to help preventpooling of cells or beads within the array chamber. Flow-enhancing ribson upper or lower surfaces of the flow cell or array chamber may be usedto control flow velocity profiles and reduce shear across the microwellopenings (i.e. to prevent cells or beads from being pulled out of themicrowells during reagent exchange and rinse steps).

In some embodiments, the instrument system may include mechanical celllysis capability as an alternative to the use of detergents or otherreagents. Sonication using a high frequency piezoelectric transducer isone example of a suitable technique.

In some embodiments, the instrument system will include temperaturecontrol functionality for the purpose of facilitating the accuracy andreproducibility of assay results, for example, cooling of the microwellarray flow cell or chamber may be advantageous for minimizing moleculardiffusion between microwells. Examples of temperature control componentsthat may be incorporated into the instrument system design include, butare not limited to, resistive heating elements, infrared light sources,Peltier heating or cooling devices, heat sinks, thermistors,thermocouples, and the like. In some embodiments of the system, thetemperature controller may provide for programmable changes intemperature over specified time intervals.

As indicated elsewhere in this disclosure, many embodiments of thedisclosed methods utilize magnetic fields for removing beads from themicrowells upon completion of the assay. In some embodiments, theinstrument system may further comprise use of magnetic fields fortransporting beads into or out of the microwell array flow cell orchamber. Examples of suitable means for providing control of magneticfields include, but are not limited to, use of electromagnets in fixedposition(s) relative to the cartridge, or the use of permanent magnetsthat are mechanically repositioned as necessary. In some embodiments ofthe instrument system, the strength of the applied magnetic field(s)will be varied by varying the amount of current applied to one or moreelectromagnets. In some embodiments of the instrument system, thestrength of the applied magnetic fields will be varied by changing theposition of one or more permanent magnets relative to the position ofthe microarray chamber(s) using, for example, stepper motor-drivenlinear actuators, servo motor-driven linear actuators, or cam shaftmechanisms. In some embodiments of the instrument system, the use ofpulsed magnetic fields may be advantageous, for example, to preventclustering of magnetic beads. In some embodiments, a magnet in closeproximity to the array or chamber may be moved, once or multiple times,between at least two positions relative to the microwell array. Motionof the magnets can serve to agitate beads within microwells, tofacilitate removal of beads from microwells, or to collect magneticbeads at a desired location.

As indicated above, in many embodiments the instrument system willinclude optical imaging and/or other spectroscopic capabilities. Suchfunctionality may be useful, for example, for inspection of themicrowell array(s) to determine whether or not the array has beenuniformly and optimally populated with cells and/or beads. Any of avariety of imaging modes may be utilized, including but not limited to,bright-field, dark-field, and fluorescence/luminescence imaging. Thechoice of imaging mode will impact the design of microwell arrays, flowcells, and cartridge chambers in that the array substrate and/oropposing wall of the flow cell or array chamber will necessarily need tobe transparent or translucent over the spectral range of interest. Insome embodiments, each microwell array may be imaged in its entiretywithin a single image. In some embodiments, a series of images may be“tiled” to create a high resolution image of the entire array. In someembodiment, a single image that represents a subsection of the array maybe used to evaluate properties, e.g., cell or bead distributions, forthe array as a whole. In some embodiments, dual wavelength excitationand emission (or multi-wavelength excitation and/or emission) imagingmay be performed. Any of a variety of light sources may be used toprovide the imaging and/or excitation light, including but not limitedto, tungsten lamps, tungsten-halogen lamps, arc lamps, lasers, lightemitting diodes (LEDs), or laser diodes. Any of a variety of imagesensors may be used for imaging purposes, including but not limited to,photodiode arrays, charge-coupled device (CCD) cameras, or CMOS imagesensors. The optical system will typically include a variety of opticalcomponents for steering, shaping, filtering, and/or focusing light beamsthrough the system. Examples of suitable optical components include, butare not limited to, lenses, mirrors, prisms, diffraction gratings,colored glass filters, narrowband interference filters, broadbandinterference filters, dichroic reflectors, optical fibers, opticalwaveguides, and the like. In some embodiments, the instrument system mayuse an optically transparent microarray substrate as a waveguide fordelivering excitation light to the microwell array. The choice ofimaging mode may also enable the use of other types of assays to be runin parallel with stochastic labeling/molecular indexing assays, forexample, the use of trypan blue live cell/dead cell assays with brightfield imaging, the use of fluorescence-based live cell/dead cell assayswith fluorescence imaging, etc. Correlation of viability data forindividual cells with the cell tag associated with each bead in theassociated microwell may provide an additional level of discriminationin analyzing the data from multiplexed, single cell assays.Alternatively, viability data in the form of statistics for multiplecells may be employed for enhancing the analytical capabilities andquality assurance of the assay.

In some embodiments, the system may comprise non-imaging and/ornon-optical capabilities for probing the microwell array. Examples ofnon-imaging and/or non-optical techniques for detecting trapped airbubbles, determining the cell and/or bead distribution over the array,etc., include but are not limited to measurements of light scattering,ultraviolet/visible/infrared absorption measurements (e.g., usingstained cells and/or beads that incorporate dyes), coherent ramanscattering, and conductance measurements (e.g., using microfabricatedarrays of electrodes in register with the microwell arrays).

System Processor and Software

In general, instrument systems designed to support the automation ofmultiplexed, single cell stochastic labeling/molecular indexing assayswill include a processor or computer, along with software to provide (i)instrument control functionality, (ii) image processing and analysiscapability, and (iii) data storage, analysis, and display functionality.

In many embodiments, the instrument system will comprise a computer (orprocessor) and computer-readable media that includes code for providinga user interface as well as manual, semi-automated, or fully-automatedcontrol of all system functions, i.e. control of the fluidics system,the temperature control system, cell and/or bead distribution functions,magnetic bead manipulation functions, and the imaging system. Examplesof fluid control functions provided by the instrument control softwareinclude, but are not limited to, volumetric fluid flow rates, fluid flowvelocities, the timing and duration for sample and bead addition,reagent addition, and rinse steps. Examples of temperature controlfunctions provided by the instrument control software include, but arenot limited to, specifying temperature set point(s) and control of thetiming, duration, and ramp rates for temperature changes. Examples ofcell and/or bead distribution functions provided by the instrumentcontrol software include, but are not limited to, control of agitationparameters such as amplitude, frequency, and duration. Examples ofmagnetic field functions provided by the instrument control softwareinclude, but are not limited to, the timing and duration of the appliedmagnetic field(s), and in the case of electromagnets, the strength ofthe magnetic field as well. Examples of imaging system control functionsprovided by the instrument control software include, but are not limitedto, autofocus capability, control of illumination and/or excitationlight exposure times and intensities, control of image acquisition rate,exposure time, and data storage options.

In some embodiments of the instrument system, the system will furthercomprise computer-readable media that includes code for providing imageprocessing and analysis capability. Examples of image processing andanalysis capability provided by the software include, but are notlimited to, manual, semi-automated, or fully-automated image exposureadjustment (e.g., white balance, contrast adjustment, signal-averagingand other noise reduction capability, etc.), automated objectidentification (i.e. for identifying cells and beads in the image),automated statistical analysis (i.e. for determining the number of cellsand/or beads identified per unit area of the microwell array, or foridentifying wells that contain more than one cell or more than onebead), and manual measurement capabilities (e.g., for measuringdistances between objects, etc.). In some embodiments, the instrumentcontrol and image processing/analysis software will be written asseparate software modules. In some embodiments, the instrument controland image processing/analysis software will be incorporated into anintegrated package. In some embodiments, the system software may provideintegrated real-time image analysis and instrument control, so that celland bead sample loading steps can be prolonged or repeated until optimalcell/bead distributions are achieved.

In some embodiments of the instrument system, the system will comprisecomputer-readable media that includes code for providing sequence dataanalysis. Examples of sequence data analysis functionality that may beprovided by the data analysis software includes, but is not limited to,(i) algorithms for determining the number of reads per gene per cell,and the number of unique transcript molecules per gene per cell, basedon the data provided by sequencing the oligonucleotide library createdby running the assay, (ii) statistical analysis of the sequencing data,e.g., principal component analysis, for predicting confidence intervalsfor determinations of the number of transcript molecules per gene percell, etc., (iii) sequence alignment capabilities for alignment of genesequence data with known reference sequences, (iv)decoding/demultiplexing of sample barcodes, cell barcodes, and molecularbarcodes, and (v) automated clustering of molecular labels to compensatefor amplification or sequencing errors.

In general, the computer or processor included in the presentlydisclosed instrument systems, as illustrated in FIG. 76, may be furtherunderstood as a logical apparatus that can read instructions from media511 and/or a network port 505, which can optionally be connected toserver 509 having fixed media 512. The system 500, such as shown in FIG.76 can include a CPU 501, disk drives 503, optional input devices suchas keyboard 515 and/or mouse 516 and optional monitor 507. Datacommunication can be achieved through the indicated communication mediumto a server at a local or a remote location. The communication mediumcan include any means of transmitting and/or receiving data. Forexample, the communication medium can be a network connection, awireless connection or an internet connection. Such a connection canprovide for communication over the World Wide Web. It is envisioned thatdata relating to the present disclosure can be transmitted over suchnetworks or connections for reception and/or review by a party 522 asillustrated in FIG. 76.

FIG. 77 is a block diagram illustrating a first example architecture ofa computer system 100 that can be used in connection with exampleembodiments of the present disclosure. As depicted in FIG. 77, theexample computer system can include a processor 102 for processinginstructions. Non-limiting examples of processors include: Intel Xeon™processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-Sv1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8Apple A4™ processor, Marvell PXA 930™ processor, or afunctionally-equivalent processor. Multiple threads of execution can beused for parallel processing. In some embodiments, multiple processorsor processors with multiple cores can also be used, whether in a singlecomputer system, in a cluster, or distributed across systems over anetwork comprising a plurality of computers, cell phones, and/orpersonal data assistant devices.

As illustrated in FIG. 77, a high speed cache 104 can be connected to,or incorporated in, the processor 102 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 102. The processor 102 is connected to a north bridge 106 by aprocessor bus 108. The north bridge 106 is connected to random accessmemory (RAM) 110 by a memory bus 112 and manages access to the RAM 110by the processor 102. The north bridge 106 is also connected to a southbridge 114 by a chipset bus 116. The south bridge 114 is, in turn,connected to a peripheral bus 118. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 118. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip.

In some embodiments, system 100 can include an accelerator card 122attached to the peripheral bus 118. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 124 and can be loadedinto RAM 110 and/or cache 104 for use by the processor. The system 100includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample embodiments of the present invention.

In this example, system 100 also includes network interface cards (NICs)120 and 121 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that can be used for distributed parallelprocessing.

FIG. 78 is a diagram showing a network 200 with a plurality of computersystems 202 a, and 202 b, a plurality of cell phones and personal dataassistants 202 c, and Network Attached Storage (NAS) 204 a, and 204 b.In example embodiments, systems 212 a, 212 b, and 212 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 214 a and 214 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 212 a, and 212 b, and cell phone and personal dataassistant systems 212 c. Computer systems 212 a, and 212 b, and cellphone and personal data assistant systems 212 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 214 a and 214 b. FIG. 78 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 79 is a block diagram of a multiprocessor computer system 300 usinga shared virtual address memory space in accordance with an exampleembodiment. The system includes a plurality of processors 302 a-f thatcan access a shared memory subsystem 304. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)306 a-f in the memory subsystem 304. Each MAP 306 a-f can comprise amemory 308 a-f and one or more field programmable gate arrays (FPGAs)310 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 310a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample embodiments. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 308a-f, allowing it to execute tasks independently of, and asynchronouslyfrom, the respective microprocessor 302 a-f. In this configuration, aMAP can feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer subsystem of the present disclosurecan be implemented using software modules executing on any of the aboveor other computer architectures and systems. In other embodiments, thefunctions of the system can be implemented partially or completely infirmware, programmable logic devices such as field programmable gatearrays (FPGAs) as referenced in FIG. 79, system on chips (SOCs),application specific integrated circuits (ASICs), or other processingand logic elements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 122 illustrated in FIG. 77.

Oligonucleotides (e.g., Molecular Barcodes)

The methods and kits disclosed herein may comprise one or moreoligonucleotides or uses thereof. The oligonucleotides may be attachedto a solid support disclosed herein. Attachment of the oligonucleotideto the solid support may occur through functional group pairs on thesolid support and the oligonucleotide. The oligonucleotide may bereferred to as a molecular bar code. The oligonucleotide may be referredto as a label (e.g., molecular label, cellular label) or tag (e.g.,sample tag).

Oligonucleotides may comprise a universal label. A universal label maybe the same for all oligonucleotides in a sample. A universal label maybe the same for oligonucleotides in a set of oligonucleotides. Auniversal label may be the same for two or more sets ofoligonucleotides. A universal label may comprise a sequence of nucleicacids that may hybridize to a sequencing primer. Sequencing primers maybe used for sequencing oligonucleotides comprising a universal label.Sequencing primers (e.g., universal sequencing primers) may comprisesequencing primers associated with high-throughput sequencing platforms.A universal label may comprise a sequence of nucleic acids that mayhybridize to a PCR primer. A universal label may comprise a sequence ofnucleic acids that may hybridize to a sequencing primer and a PCRprimer. The sequence of nucleic acids of the universal label that mayhybridize to a sequencing and/or PCR primer may be referred to as aprimer binding site. A universal label may comprise a sequence that maybe used to initiate transcription of the oligonucleotide. A universallabel may comprise a sequence that may be used for extension of theoligonucleotide or a region within the oligonucleotide. A universallabel may be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50 or more nucleotides in length. A universal label may comprise atleast about 10 nucleotides. A universal label may be at most about 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides inlength.

Oligonucleotides may comprise a cellular label. A cellular label maycomprise a nucleic acid sequence that may provide information for whichcell the oligonucleotide is contacted to (e.g., determining whichnucleic acid originated from which cell). At least 60%, 70%, 80%, 85%,90%, 95%, 97%, 99% or 100% of oligonucleotides on the same solid supportmay comprise the same cellular label. At least 60% of oligonucleotideson the same solid support may comprise the same cellular label. At least95% of oligonucleotides on the same solid support may comprise the samecellular label. All the oligonucleotides on a same solid support maycomprise the same cellular label. The cellular label of theoligonucleotides on a first solid support may be different than thecellular labels of the oligonucleotides on the second solid support.

A cellular label may be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50 or more nucleotides in length. A cellular label maybe at most about 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12,10, 9, 8, 7, 6, 5, 4 or fewer or more nucleotides in length. A cellularlabel may comprise between about 5 to about 200 nucleotides. A cellularlabel may comprise between about 10 to about 150 nucleotides. A cellularlabel may comprise between about 20 to about 125 nucleotides in length.

Oligonucleotides may comprise a molecular label. A molecular label maycomprise a nucleic acid sequence that may provide identifyinginformation for the specific nucleic acid species hybridized to theoligonucleotide. Oligonucleotides conjugated to a same solid support maycomprise different molecular labels. In this way, the molecular labelmay distinguish the types of target nucleic acids (e.g., genes), thathybridize to the different oligonucleotides. A molecular label may be atleast about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or morenucleotides in length. A molecular label may be at most about 300, 200,100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 orfewer nucleotides in length.

Oligonucleotides may comprise a sample label (e.g., sample index). Asample label may comprise a nucleic acid sequence that may provideinformation about from where a target nucleic acid originated. Forexample, a sample label may be different on different solid supportsused in different experiments. A sample label may be at least about 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides inlength. A sample label may be at most about 300, 200, 100, 90, 80, 70,60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or fewer nucleotides inlength.

An oligonucleotide may comprise a universal label, a cellular label, amolecular label and a sample label, or any combination thereof. Incombination, the sample label may be used to distinguish target nucleicacids between samples, the cellular label may be used to distinguishtarget nucleic acids from different cells in the sample, the molecularlabel may be used to distinguish the different target nucleic acids inthe cell (e.g., different copies of the same target nucleic acid), andthe universal label may be used to amplify and sequence the targetnucleic acids.

A universal label, a molecular label, a cellular label, linker labeland/or a sample label may comprise a random sequence of nucleotides. Arandom sequence of nucleotides may be computer generated. A randomsequence of nucleotides may have no pattern associated with it. Auniversal label, a molecular label, a cellular label, linker labeland/or a sample label may comprise a non-random (e.g., the nucleotidescomprise a pattern) sequence of nucleotides. Sequences of the universallabel, a molecular label, a cellular label, linker label and/or a samplelabel may be commercially available sequences. Sequences of theuniversal label, a molecular label, a cellular label, linker labeland/or a sample label may be comprise randomer sequences. Randomersequences may refer to oligonucleotide sequences composed of allpossible sequences for a given length of the randomer. Alternatively, oradditionally, a universal label, a molecular label, a cellular label,linker label and/or a sample label may comprise a predetermined sequenceof nucleotides.

FIG. 1 shows an exemplary oligonucleotide of the disclosure comprising auniversal label, a cellular label and a molecular label.

FIG. 3 shows an exemplary oligonucleotide coupled solid supportcomprising a solid support (301) coupled to an oligonucleotide (312).The oligonucleotide (312) comprises a chemical group (5′ amine, 302), auniversal label (303), a cellular label (311), a molecular label(Molecular BC, 311), and a target binding region (oligodT, 310). In thisschematic, the cellular label (311) comprises a first cell label (CLPart 1, 304), a first linker (Linked, 305), a second cell label (CL Part2, 306), a second linker (Linker2, 307), a third cell label (CL Part 3,308). The cellular label (311) is common for each oligonucleotide on thesolid support. The cellular labels (311) for two or more beads may bedifferent. The cellular labels (311) for two or more beads may differ bythe cell labels (e.g., CL Part 1 (304), CL Part 2 (306), CL Part 3(308)). The cellular labels (311) for two or more beads may differ bythe first cell label (304), second cell label (306), third cell label(308), or a combination thereof. The first and second linkers (303, 305)of the cellular labels (311) may be identical for two or moreoligonucleotide coupled solid supports. The universal label (303) may beidentical for two or more oligonucleotide coupled solid supports. Theuniversal label (303) may be identical for two or more oligonucleotideson the same solid support. The molecular label (311) may be differentfor at least two or more oligonucleotides on the solid support. Thesolid support may comprise 100 or more oligonucleotides. The solidsupport may comprise 1000 or more oligonucleotides. The solid supportmay comprise 10000 or more oligonucleotides. The solid support maycomprise 100000 or more oligonucleotides.

In addition to a universal label, a cellular label, and a molecularlabel, an oligonucleotide may comprise a target binding region. A targetbinding region may comprise a nucleic acid sequence that may bind to atarget nucleic acid (e.g., a cellular nucleic acid to be analyzed). Atarget binding region may be a gene specific sequence. For example, atarget binding region may comprise a nucleic acid sequence that mayattach (e.g., hybridize) to a specific location of a specific targetnucleic acid. A target binding region may comprise a non-specific targetnucleic acid sequence. A non-specific target nucleic acid sequence mayrefer to a sequence that may bind to multiple target nucleic acids,independent of the specific sequence of the target nucleic acid. Forexample, target binding region may comprise a random multimer sequenceor an oligo dT sequence (e.g., a stretch of thymidine nucleotides thatmay hybridize to a poly-adenylation tail on mRNAs). A random multimersequence can be, for example, a random dimer, trimer, quatramer,pentamer, hexamer, septamer, octamer, nonamer, decamer, or highermultimer sequence of any length. A target binding region may be at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides inlength. A target binding region may be at most about 5, 10, 15, 20, 25,30, 35, 40, 45, 50 or more nucleotides in length.

An oligonucleotide may comprise a plurality of labels. For example anoligonucleotide may comprise at least about 1, 2, 3, 4, 5, 6, 7, or 8 ormore universal labels. An oligonucleotide may comprise at most about 1,2, 3, 4, 5, 6, 7, or 8 or more universal labels. An oligonucleotide maycomprise at least about 1, 2, 3, 4, 5, 6, 7, or 8 or more cellularlabels. An oligonucleotide may comprise at most about 1, 2, 3, 4, 5, 6,7, or 8 or more cellular labels. An oligonucleotide may comprise atleast about 1, 2, 3, 4, 5, 6, 7, or 8 or more molecular labels. Anoligonucleotide may comprise at most about 1, 2, 3, 4, 5, 6, 7, or 8 ormore molecular labels. An oligonucleotide may comprise at least about 1,2, 3, 4, 5, 6, 7, or 8 or more sample labels. An oligonucleotide maycomprise at most about 1, 2, 3, 4, 5, 6, 7, or 8 or more sample labels.An oligonucleotide may comprise at least about 1, 2, 3, 4, 5, 6, 7, or 8or more target binding regions. An oligonucleotide may comprise at mostabout 1, 2, 3, 4, 5, 6, 7, or 8 or more target binding regions.

When an oligonucleotide comprises more than one of a type of label(e.g., more than one cellular label or more than one molecular label),the labels may be interspersed with a linker label sequence. A linkerlabel sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50 or more nucleotides in length. A linker label sequence may be at mostabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides inlength. In some instances, a linker label sequence is 12 nucleotides inlength. A linker label sequence may be used to facilitate the synthesisof the oligonucleotide, such as diagrammed in FIG. 2A.

The number of oligonucleotides conjugated to a solid support may be 1,2, 3, 4, 5, 6, 7, 8, 9, or 10-fold more than the number of targetnucleic acids in a cell. In some instances, at least 10, 20, 30, 40, 50,60, 70, 80, 90 or 100% of the oligonucleotides are bound by a targetnucleic acid. In some instances, at most 10, 20, 30, 40, 50, 60, 70, 80,90 or 100% of the oligonucleotides are bound by a target nucleic acid.In some instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 or more different target nucleic acids arecaptured by the oligonucleotides on a solid support. In some instances,at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or100 or more different target nucleic acids are captured by theoligonucleotides on a solid support.

A polymer may comprise additional solid supports. For example, a polymermay be dotted with beads. The beads may be spatially located atdifferent regions of the polymer. The beads or supports comprisingoligonucleotides of the disclosure may be spatially addressed. The beadsor supports may comprise a barcode corresponding to a spatial address onthe polymer. For example, each bead or support of a plurality of beadsor supports may comprise barcode that corresponds to a position on apolymer, such as a position on an array or a particular microwall of aplurality of microwells. The spatial address can be decoded to determinethe location from which a bead or support was positioned. For example, aspatial address, such as a barcode, can be decoded by hybridization ofan oligonucleotide to the barcode or by sequencing the barcode.Alternatively, beads or supports can bear other types of barcodes, suchas graphical features, chemical groups, colors, fluorescence, orcombinations any combination thereof, for spatial address decodingpurposes.

The methods and kits disclosed herein may comprise one or more sets ofmolecular barcodes. One or more molecular barcodes may comprise a sampleindex region and a label region. Two or more molecular barcodes of a setof molecular barcodes may comprise the same sample index region and twoor more different label regions. Two or more molecular barcodes of twoor more sets of molecular barcodes may comprise two or more differentsample index regions. Two or more molecular barcodes from a set ofmolecular barcodes may comprise different label regions. Two or moremolecular barcodes of two or more sets of molecular barcodes maycomprise the same label region. Molecular barcodes from two or more setsof molecular barcodes may differ by their sample index regions.Molecular barcodes from two or more sets of molecular barcodes may besimilar based on their label regions.

The molecular barcodes may further comprise a target specific region, anadapter region, a universal PCR region, a target specific region or anycombination thereof. The molecular barcode may comprise a universal PCRregion and a target specific region. The molecular barcode may compriseone or more secondary structures. The molecular barcode may comprise ahairpin structure. The molecular barcode may comprise a target specificregion and a cleavable stem.

The methods and kits disclosed herein may comprise one or more sets ofsample tags. One or more sample tags may comprise a sample index region.One or more sample tags may comprise a sample index region. Two or moresample tags of a set of sample tags may comprise the same sample indexregion. Two or more sample tags of two or more sets of sample tags maycomprise two or more different sample index regions.

The sample tags may further comprise a target specific region, anadapter region, a universal PCR region, a target specific region or anycombination thereof. The sample tag may comprise a universal PCR regionand a target specific region. The sample tag may comprise one or moresecondary structures. The sample tag may comprise a hairpin structure.The sample tag may comprise a target specific region and a cleavablestem.

The methods and kits disclosed herein may comprise one or more sets of,molecular identifier labels. One or more molecular identifier labels maycomprise a label region. One or more molecular identifier labels maycomprise a label region. Two or more molecular identifier labels of aset of molecular identifier labels may comprise two or more differentlabel regions. Two or more molecular identifier labels of two or moresets of molecular identifier labels may comprise two or more identicallabel regions. The molecular identifier labels may further comprise atarget specific region, an adapter region, a universal PCR region, atarget specific region or any combination thereof. The molecularidentifier label may comprise a universal PCR region and a targetspecific region. The molecular identifier label may comprise one or moresecondary structures. The molecular identifier label may comprise ahairpin structure. The molecular identifier label may comprise a targetspecific region and a cleavable stem.

The molecular barcode, sample tag or molecular identifier label maycomprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 nucleotides or base pairs. In anotherexample, the sample tag or molecular identifier label comprises at leastabout 1500, 2,000; 2500, 3,000; 3500, 4,000; 4500, 5,000; 5500, 6,000;6500, 7,000; 7500, 8,000; 8500, 9,000; 9500, or 10,000 nucleotides orbase pairs.

The molecular barcodes, sample tags or molecular identifier labels maybe multimers, e.g., random multimers. A multimer sequence can be, forexample, a non-random or random dimer, trimer, quatramer, pentamer,hexamer, septamer, octamer, nonamer, decamer, or higher multimersequence of any length. The tags may be randomly generated from a set ofmononucleotides. The tags may be assembled by randomly incorporatingmononucleotides.

The molecular barcodes, sample tags or molecular identifier labels mayalso be assembled without randomness, to generate a library of differenttags which are not randomly generated but which includes sufficientnumbers of different tags to practice the methods.

In some embodiments a molecular barcode, sample tag or molecularidentifier label may comprise a cutback in a target nucleic acid. Thecutback may be, for example, an enzymatic digestion of one or both endsof a target nucleic acid. The cutback may be used in conjunction withthe addition of added molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label). The combination of the cutback and theadded tags may contain information related to the particular startingmolecule. By adding a random cutback to the molecular barcode, sampletag or molecular identifier label, a smaller diversity of the added tagsmay be necessary for counting the number of target nucleic acids whendetection allows a determination of both the random cutback and theadded oligonucleotides.

The molecular barcode, sample tag or molecular identifier label maycomprise a target specific region. The target specific region maycomprise a sequence that is complementary to the molecule. In someinstances, the molecule is an mRNA molecule and the target specificregion comprises an oligodT sequence that is complementary to the polyAtail of the mRNA molecule. The target specific region may also act as aprimer for DNA and/or RNA synthesis. For example, the oligodT sequenceof the target specific region may act as a primer for first strandsynthesis of a cDNA copy of the mRNA molecule. Alternatively, the targetspecific region comprises a sequence that is complementary to anyportion of the molecule. In other instances, the target specific regioncomprises a random sequence that may be hybridized or ligated to themolecule. The target specific region may enable attachment of the sampletag or molecular identifier label to the molecule. Attachment of thesample tag or molecular identifier label may occur by any of the methodsdisclosed herein (e.g., hybridization, ligation). In some instances, thetarget specific region comprises a sequence that is recognized by one ormore restriction enzymes. The target specific region may comprise atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 nucleotides or base pairs. In another example,the target specific region comprises at least about 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, or 10000 nucleotides or base pairs. Preferably, the targetspecific region comprises at least about 5-10, 10-15, 10-20, 10-30,15-30, or 20-30 nucleotides or base pairs.

In some instances, the target specific region is specific for aparticular gene or gene product. For example, the target specific regioncomprises a sequence complementary to a region of a p53 gene or geneproduct. Therefore, the sample tags and molecular identifier labels mayonly attach to molecules comprising the p53-specific sequence.Alternatively, the target specific region is specific for a plurality ofdifferent genes or gene products. For example, the target specificregion comprises an oligodT sequence. Therefore, the sample tags andmolecular identifier labels may attach to any molecule comprising apolyA sequence. In another example, the target specific region comprisesa random sequence that is complementary to a plurality of differentgenes or gene products. Thus, the sample tag or molecular identifierlabel may attach to any molecule with a sequence that is complementaryto the target specific region. In other instances, the target specificregion comprises a restriction site overhang (e.g., EcoRI sticky-endoverhang). The sample tag or molecular identifier label may ligate toany molecule comprising a sequence complementary to the restriction siteoverhang.

In some instances, the target specific region is specific for aparticular microRNA or microRNA product. For example, the targetspecific region comprises a sequence complementary to a region of aspecific microRNA or microRNA product. For example, the target specificregions comprise sequences complementary to regions of a specific panelof microRNAs or panel of microRNA products. Therefore, the sample tagsand molecular identifier labels may only attach to molecules comprisingthe micoRNA-specific sequence. Alternatively, the target specific regionis specific for a plurality of different micoRNAs or micoRNA products.For example, the target specific region comprises a sequencecomplimentary to a region comprised in two or more microRNAs, such as apanel of microRNAs containing a common sequence. Therefore, the sampletags and molecular identifier labels may attach to any moleculecomprising the common microRNA sequence. In another example, the targetspecific region comprises a random sequence that is complementary to aplurality of different microRNAs or microRNA products. Thus, the sampletag or molecular identifier label may attach to any microRNA moleculewith a sequence that is complementary to the target specific region. Inother instances, the target specific region comprises a restriction siteoverhang (e.g., EcoRI sticky-end overhang). The sample tag or molecularidentifier label may ligate to any microRNA molecule comprising asequence complementary to the restriction site overhang.

The molecular barcode or molecular identifier label disclosed hereinoften comprises a label region. The label region may be used to uniquelyidentify occurrences of target species thereby marking each species withan identifier that may be used to distinguish between two otherwiseidentical or nearly identical targets. The label region of the pluralityof sample tags and molecular identifier labels may comprise a collectionof different semiconductor nanocrystals, metal compounds, peptides,oligonucleotides, antibodies, small molecules, isotopes, particles orstructures having different shapes, colors, barcodes or diffractionpatterns associated therewith or embedded therein, strings of numbers,random fragments of proteins or nucleic acids, different isotopes, orany combination thereof. The label region may comprise a degenerativesequence. The label region may comprise at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotidesor base pairs. In another example, the label region comprises at leastabout 1500; 2,000; 2500, 3,000; 3500, 4,000; 4500, 5,000; 5500, 6,000;6500, 7,000; 7500, 8,000; 8500, 9,000; 9500, or 10,000 nucleotides orbase pairs. Preferably, the label region comprises at least about 10-30,15-40, or 20-50 nucleotides or base pairs.

In some instances, the molecular barcode, sample tag or molecularidentifier label comprises a universal primer binding site. Theuniversal primer binding site allows the attachment of a universalprimer to the labeled-molecule and/or labeled-amplicon. Universalprimers are well known in the art and include, but are not limited to,−47F (M13F), alfaMF, AOX3′, AOX5′, BGH_r, CMV_−30, CMV_−50, CVM_f,LACrmt, lamgda gt10F, lambda gt 10R, lambda gt11F, lambda gt11R, M13rev, M13Forward(−20), M13Reverse, male, p10SEQP_pQE, pA_−120, pet_4,pGAP Forward, pGL_RVpr3, pGLpr2_R, pKLAC1_4, pQE_FS, pQE_RS, puc_U1,puc_U2, revers_A, seq_IRES_tam, seq_IRES_zpet, seq_ori, seq_PCR,seq_IRES−, seq_pIRES+, seq_pSecTag, seq_pSecTag+, seq_retro+PSI, SP6,T3-prom, T7-prom, and T7-term Inv. Attachment of the universal primer tothe universal primer binding site may be used for amplification,detection, and/or sequencing of the labeled-molecule and/orlabeled-amplicon. The universal primer binding site may comprise atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 nucleotides or base pairs. In another example,the universal primer binding site comprises at least about 1500; 2,000;2500, 3,000; 3500, 4,000; 4500, 5,000; 5500, 6,000; 6500, 7,000; 7500,8,000; 8500, 9,000; 9500, or 10,000 nucleotides or base pairs.Preferably, the universal primer binding site comprises 10-30nucleotides or base pairs.

The molecular barcode, sample tag or molecular identifier label maycomprise an adapter region. The adapter region may enable hybridizationof one or more probes. The adapter region may enable hybridization ofone or more HCR probes.

The molecular barcode, sample tag or molecular identifier label maycomprise one or more detectable labels.

The molecular barcode, sample tag or molecular identifier label may actas an initiator for a hybridization chain reaction (HCR). The adapterregion of the sample tag or molecular identifier label may act as aninitiation for HCR. The universal primer binding site may act as aninitiator for HCR.

In some instances, the molecular barcode, sample tag or molecularidentifier label is single-stranded. In other instances, the molecularbarcode, sample tag or molecular identifier label is double-stranded.The molecular barcode, sample tag or molecular identifier label may belinear. Alternatively, the molecular barcode, sample tag or molecularidentifier label comprises a secondary structure. As used herein,“secondary structure” includes tertiary, quaternary, etc. . . .structures. In some instances, the secondary structure is a hairpin, astem-loop structure, an internal loop, a bulge loop, a branchedstructure or a pseudoknot, multiple stem loop structures, cloverleaftype structures or any three dimensional structure. In some instances,the secondary structure is a hairpin. The hairpin may comprise anoverhang sequence. The overhang sequence of the hairpin may act as aprimer for a polymerase chain reaction and/or reverse transcriptionreaction. The overhang sequence comprises a sequence that iscomplementary to the molecule to which the sample tag or molecularidentifier label is attached and the overhang sequence hybridizes to themolecule. The overhang sequence may be ligated to the molecule and actsas a template for a polymerase chain reaction and/or reversetranscription reaction. In some embodiments, molecular barcode, thesample tag, or molecular identifier label comprises nucleic acids and/orsynthetic nucleic acids and/or modified nucleic acids.

In some instances, the plurality of molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) comprises at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 different molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label). In other instances, theplurality of molecular barcodes, sample tags (e.g., sample index region,sample label), cellular label, and molecular identifier labels (e.g.,molecular label) comprises at least about 200; 300; 400; 500; 600; 700;800; 900; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000;or 10000 different molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label). Alternatively; the plurality of molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)comprises at least about 20,000; 30,000; 40,000; 50,000; 60,000; 70,000;80,000; 90,000; or 100,000 different molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label).

The number of molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label) in the plurality of molecular barcodes, sampletags (e.g., sample index region, sample label), cellular label, andmolecular identifier labels (e.g., molecular label) is often in excessof the number of molecules to be labeled. In some instances, the numberof molecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) in the plurality of molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label) is at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than thenumber of molecules to be labeled.

The number of different molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label) in the plurality of molecular barcodes,sample tags (e.g., sample index region, sample label), cellular label,and molecular identifier labels (e.g., molecular label) is often inexcess of the number of different molecules to be labeled. In someinstances, the number of different molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) in the plurality of molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the number ofdifferent molecules to be labeled.

In some instances, stochastic labeling of a molecule comprises aplurality of molecular barcodes, sample tags (e.g., sample index region,sample label), cellular label, and molecular identifier labels (e.g.,molecular label), wherein the concentration of the different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)in the plurality of molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label) is the same. In such instances, the plurality ofmolecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) comprises equal numbers of each different molecular barcode,sample tag or molecular identifier label.

In some instances, stochastic labeling of a molecule comprises aplurality of molecular barcodes, sample tags (e.g., sample index region,sample label), cellular label, and molecular identifier labels (e.g.,molecular label), wherein the concentration of the different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)in the plurality of molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label) is different. In such instances, the pluralityof molecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) comprises different numbers of each different molecular barcode,sample tag or molecular identifier label.

In some instances, some molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label) are present at higher concentrations thanother molecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) in the plurality of molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label). In some instances, stochastic labelingwith different concentrations of molecular barcodes, sample tags (e.g.,sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) extends the sample measurementdynamic range without increasing the number of different labels used.For example, consider stochastically labeling 3 nucleic acid samplemolecules with 10 different molecular barcodes, sample tags (e.g.,sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) all at equal concentration. Weexpect to observe 3 different labels. Now instead of 3 nucleic acidmolecules, consider 30 nucleic acid molecules, and we expect to observeall 10 labels. In contrast, if we still used 10 different stochasticlabels and alter the relative ratios of the labels to 1:2:3:4 . . . 10,then with 3 nucleic acid molecules, we would expect to observe between1-3 labels, but with 30 molecules we would expect to observe onlyapproximately 5 labels thus extending the range of measurement with thesame number of stochastic labels.

The relative ratios of the different molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) in the plurality of molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)may be 1:X, where X is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100.Alternatively, the relative ratios of “n” different molecular barcodes,sample tags (e.g., sample index region, sample label), cellular label,and molecular identifier labels (e.g., molecular label) in the pluralityof molecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) is 1:A:B:C: . . . Zn, where A, B, C . . . Zn is at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100.

In some instances, the concentration of two or more different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)in the plurality of molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label) is the same. For “n” different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label),the concentration of at least 2, 3, 4, . . . n different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)is the same. Alternatively, the concentration of two or more differentmolecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) in the plurality of molecular barcodes, sample tags (e.g., sampleindex region, sample label), cellular label, and molecular identifierlabels (e.g., molecular label) is different. For “n” different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label),the concentration of at least 2, 3, 4, . . . n different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)is different. In some instances, for “n” different molecular barcodes,sample tags (e.g., sample index region, sample label), cellular label,and molecular identifier labels (e.g., molecular label), the differencein concentration for at least 2, 3, 4, . . . n different molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25,1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000-fold.

In some instances, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, or 100% of the different molecular barcodes,sample tags (e.g., sample index region, sample label), cellular label,and molecular identifier labels (e.g., molecular label) in the pluralityof molecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) have the same concentration. Alternatively, at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of thedifferent molecular barcodes, sample tags (e.g., sample index region,sample label), cellular label, and molecular identifier labels (e.g.,molecular label) in the plurality of molecular barcodes, sample tags(e.g., sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) have a differentconcentration.

As shown in FIG. 65, molecular barcodes (1004) may be synthesizedseparately. The molecular barcodes (1004) may comprise a universal PCRregion (1001), one or more identifier regions (1002), and a targetspecific region. The one or more identifier regions may comprise asample index region, label region, or a combination thereof. The one ormore identifier regions may be adjacent. The one or more identifierregions may be non-adjacent. The individual molecular barcodes may bepooled to produce a plurality of molecular barcodes (1005) comprising aplurality of different identifier regions. Sample tags may besynthesized in a similar manner as depicted in FIG. 65, wherein the oneor more identifier regions comprise a sample index region. Molecularidentifier labels may be synthesized in a similar manner as depicted inFIG. 65, wherein the one or more identifier regions comprises a labelregion.

The target specific region may be ligated to the identifier region toproduce a molecular barcode comprising a target specific region. 5′ and3′ exonucleases may be added to the reaction to remove non-ligatedproducts. The molecular barcode may comprise the universal primerbinding site, label region and target specific region and may beresistant to 5′ and 3′ exonucleases. As used herein, the terms“universal primer binding site” and “universal PCR region” may be usedinterchangeably and refer to a sequence that can be used to prime anamplification reaction. The 3′ phosphate group from the ligatedidentifier region may be removed to produce a molecular barcode withouta 3′ phosphate group. The 3′ phosphate group may be removedenzymatically. For example, a T4 polynucleotide kinase may be used toremove the 3′ phosphate group.

Another method of synthesizing molecular barcodes is depicted in FIG.66A. As shown in FIG. 66A, a molecular barcode (1128) may be synthesizedby ligating two or more oligonucleotide fragments (1121 and 1127). Oneoligonucleotide fragment (1121) may comprise a universal primer bindingsite (1122), identifier region (1123) and a first splint (1123). Theother oligonucleotide fragment (1128) may comprise a second splint(1125) and a target specific region (1126). A ligase (e.g., T4 DNAligase) may be used to join the two oligonucleotide fragments (1121 and1127) to produce a molecular barcode (1128). Double stranded ligation ofthe first splint (1124) and second splint (1125) may produce a molecularbarcode (1128) with a bridge splint (1129).

An alternative method of synthesizing a molecular barcode by ligatingtwo oligonucleotide fragments is depicted in FIG. 66B. As shown in FIG.66B, a molecular barcode (1158) is synthesized by ligating twooligonucleotide fragments (1150 and 1158). One oligonucleotide fragment(1150) may comprise a universal primer binding site (1151), one or moreidentifier region (1152), and a ligation sequence (1153). The otheroligonucleotide fragment (1158) may comprise a ligation sequence (1154)that is complementary to the ligation sequence (1153) of the firstoligonucleotide fragment (1150), a complement of a target specificregion (1155), and a label (1156). The oligonucleotide fragment (1159)may also comprise a 3′ phosphate which prevents extension of theoligonucleotide fragment. As shown in Step 1 of FIG. 66B, the ligationsequences (1153 and 1154) of the two oligonucleotide fragments mayanneal and a polymerase may be used to extend the 3′ end of the firstoligonucleotide fragment (1150) to produce molecular barcode (1158). Themolecular barcode (1158) may comprise a universal primer binding site(1151), one or more identifier regions (1152), ligation sequence (1153),and a target specific sequence (1157). The target specific sequence(1157) of the molecular barcode (1158) may be the complement of thecomplement of the target specific region (1155) of the secondoligonucleotide fragment (1159). The oligonucleotide fragment comprisingthe label (1156) may be removed from the molecular barcode (1158). Forexample, the label (1156) may comprise biotin and oligonucleotidefragments (1159) comprising the biotin label (1156) may be removed viastreptavidin capture. In another example, the label (1156) may comprisea 5′ phosphate and oligonucleotide fragments (1159) comprising the 5′phosphate (1156) may be removed via an exonuclease (e.g., Lambdaexonuclease).

As depicted in FIG. 66C, a first oligonucleotide fragment (1170)comprising a universal primer binding site (1171), one or moreidentifier regions (1172), a first ligation sequence (1173) is annealedto a second oligonucleotide fragment (1176) comprising a second ligationsequence (1174) and an RNA complement of the target sequence (1175).Step 1 may comprise annealing the first and second ligation sequences(1173 and 1174) followed by reverse transcription of the RNA complementof the target sequence (1175) to produce molecular barcode (1177)comprising a universal primer binding site (1171), one or moreidentifier regions (1172), a first ligation sequence (1173), and atarget specific region (1178). The oligonucleotide fragments comprisingthe RNA complement of the target sequence may be selectively degraded byRNAse treatment.

The sequences of the molecular barcodes, sample tags (e.g., sample indexregion, sample label), cellular label, and molecular identifier labels(e.g., molecular label) may be optimized to minimize dimerization ofmolecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel). The molecular barcode, sample tag or molecular identifier labeldimer may be amplified and result in the formation of an ampliconcomprising two universal primer binding sites on each end of theamplicon and a target specific region and a unique identifier region.Because the concentration of the molecular barcodes, sample tags (e.g.,sample index region, sample label), cellular label, and molecularidentifier labels (e.g., molecular label) are far greater that thenumber of DNA templates, these molecular barcode, sample tag ormolecular identifier label dimers may outcompete the labeled DNAmolecules in an amplification reaction. Unamplified DNAs lead to falsenegatives, and amplified molecular barcode, sample tag or molecularidentifier label dimers lead to high false positives. Thus, themolecular barcodes, sample tags (e.g., sample index region, samplelabel), cellular label, and molecular identifier labels (e.g., molecularlabel) may be optimized to minimize molecular barcode, sample tag ormolecular identifier label dimer formation. Alternatively, molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)that dimerize are discarded, thereby eliminating molecular barcode,sample tag or molecular identifier label dimer formation.

Alternatively, molecular barcode, sample tag or molecular identifierlabel dimer formation may be eliminated or reduced by incorporating oneor more modifications into the molecular barcode, sample tag ormolecular identifier label sequence. A molecular barcode, sample tag ormolecular identifier label comprising a universal primer binding site,unique identifier region, and target specific region comprising uracilsand a 3′ phosphate group is annealed to a target nucleic acid. Thetarget nucleic acid may be a restriction endonuclease digested fragment.The restriction endonuclease may recognize the recognition site. PCRamplification may comprise one or more forward primers and one or morereverse primers. PCR amplification may comprise nested PCR with aforward primer specific for the universal primer binding site of themolecular barcode, sample tag or molecular identifier label and aforward primer specific for the target specific region of the molecularbarcode, sample tag or molecular identifier label and reverse primersthat are specific for the target nucleic acid. The target nucleic acidmay be amplified using a Pfu DNA polymerase, which cannot amplifytemplate comprising one or more uracils. Thus, any dimerized molecularbarcodes, sample tags (e.g., sample index region, sample label),cellular label, and molecular identifier labels (e.g., molecular label)cannot be amplified by Pfu DNA polymerase.

Methods to Synthesize Oligonucleotides (e.g., Molecular Barcodes)

An oligonucleotide may be synthesized. An oligonucleotide may besynthesized, for example, by coupling (e.g., by1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) of a 5′ amino group onthe oligonucleotide to the carboxyl group of the functionalized solidsupport.

Uncoupled oligonucleotides may be removed from the reaction mixture bymultiple washes. The solid supports may be split into wells (e.g., 96wells). Each solid support may be split into a different well.Oligonucleotide synthesis may be performed using the split/pool methodof synthesis. The split/pool method may utilize a pool of solid supportscomprising reactive moieties (e.g., oligonucleotides to be synthesized).This pool may be split into a number of individual pools of solidsupports. Each pool may be subjected to a first reaction that may resultin a different modification to the solid supports in each of the pools(e.g., a different nucleic acid sequence added to the oligonucleotide).After the reaction, the pools of solid supports may be combined, mixed,and split again. Each split pool may be subjected to a second reactionor randomization that again is different for each of the pools. Theprocess may be continued until a library of target compounds is formed.

Using split/pool synthesis, the nucleic acid sequence to be added to theoligonucleotide may be incorporated by primer extension (e.g., Klenowextension). The nucleic acid sequence to be added to the oligonucleotidemay be referred to as a primer fragment. Each primer fragment for eachindividual pool may comprise a different sequence (e.g., either in thecellular label, the molecular label, the sample label, or anycombination thereof). The primer fragment may comprise a sequence thatmay hybridize to the linker label sequence of the oligonucleotide (e.g.,the oligonucleotide coupled to the solid support). The primer fragmentmay further comprise a second cell label and a second linker labelsequence. Primer extension may be used to introduce the second celllabel sequence and the second linker label sequence onto theoligonucleotide coupled to the solid support (See FIG. 2B). After primerextension incorporates the new sequences, the solid supports may becombined. The combined solid supports may be heated to denature theenzyme. The combined solid supports may be heated to disrupthybridization. The combined solid supports may be split into wellsagain. The process may be repeated to add additional sequences to thesolid support-conjugated oligonucleotide.

The split/pool process may lead to the creation of at least about 1000,10000, 100000, 500000, or 1000000 or more different oligonucleotides.The process may lead to the creation of at most about 1000, 10000,100000, 500000, or 1000000 or more different oligonucleotides.

Split pool synthesis may comprise chemical synthesis. Differentoligonucleotides may be synthesized using DMT chemistry on solidsupports in individual reactions, then pooled into reactions forsynthesis. The split/pool process may be repeated 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more times. The split/pool process may be repeated 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more times. The split/pool process may berepeated 2 or more times. The split/pool process may be repeated 3 ormore times. The split/pool process may be repeated 5 or more times. Thesplit/pool process may be repeated 10 or more times.

Further disclosed herein are methods of producing one or more sets oflabeled beads (e.g., oligonucleotide conjugated beads). The method ofproducing the one or more sets of labeled beads may comprise attachingone or more nucleic acids to one or more beads, thereby producing one ormore sets of labeled beads. The one or more nucleic acids may compriseone or more molecular barcodes. The one or more nucleic acids maycomprise one or more sample tags (e.g., sample labels, sample indexregions). The one or more nucleic acids may comprise one or morecellular labels. The one or more nucleic acids may comprise one or moremolecular identifier labels (e.g., molecular labels). The one or morenucleic acids may comprise a) a primer region; b) a sample index region;and c) a linker or adaptor region. The one or more nucleic acids maycomprise a) a primer region; b) a label region (e.g., molecular label);and c) a linker or adaptor region. The one or more nucleic acids maycomprise a) a sample index region (e.g., sample tag); and b) a labelregion (e.g., molecular label). The one or more nucleic acids maycomprise a) a sample index region; and b) a cellular label. The one ormore nucleic acids may comprise a) a cellular label; and b) a molecularlabel. The one or more nucleic acids may comprise a) a sample indexregion; b) cellular label; and c) a molecular label. The one or morenucleic acids may further comprise a primer region. The one or morenucleic acids may further comprise a target specific region. The one ormore nucleic acids may further comprise a linker region. The one or morenucleic acids may further comprise an adaptor region. The one or morenucleic acids may further comprise a sample index region. The one ormore nucleic acids may further comprise a label region.

Alternatively, the method comprises: a) depositing a plurality of firstnucleic acids into a plurality of wells, wherein two or more differentwells of the plurality of wells may comprise two or more differentnucleic acids of the plurality of nucleic acids; b) contacting one ormore wells of the plurality of wells with one or fewer beads to producea plurality of single label beads, wherein a single label bead of theplurality of first labeled beads comprises a bead attached to a nucleicacid of the plurality of first nucleic acids; c) pooling the pluralityof first labeled beads from the plurality of wells to produce a pool offirst labeled beads; d) distributing the pool of first labeled beads toa subsequent plurality of wells, wherein two or more wells of thesubsequent plurality of wells comprise two or more different nucleicacids of a plurality of subsequent nucleic acids; and e) attaching oneor more nucleic acids of the plurality of subsequent nucleic acids toone or more first labeled beads to produce a plurality of uniquelylabeled beads.

Libraries

Disclosed herein are methods of producing molecular libraries. Themethod may comprise: (a) stochastically labeling two or more moleculesfrom two or more samples to produce labeled molecules, wherein thelabeled molecules comprise (i) a molecule region based on or derivedfrom the two or more molecules, (ii) a sample index region for use indifferentiating two or more molecules from two or more samples; and(iii) a label region for use in differentiating two or more moleculesfrom a single sample. Stochastic labeling may comprise the use of one ormore sets of molecular barcodes. Stochastic labeling may comprise theuse of one or more sets of sample tags. Stochastic labeling may comprisethe use of one or more sets of molecular identifier labels.

Stochastically labeling the two or more molecules may comprisecontacting the two or more samples with a plurality of sample tags andthe plurality of molecule specific labels to produce the plurality oflabeled nucleic acids. The contacting can be random. The method mayfurther comprise amplifying one or more of the labeled molecules,thereby producing an enriched population of labeled molecules of thelibrary. The method may further comprise conducting one or more assayson the two or more molecules from the two or more samples. The methodmay further comprise conducting one or more pull-down assays.

The method of producing a labeled nucleic acid library may furthercomprise adding one or more controls to the two or more of samples. Theone or more controls may be stochastically labeled to produce labeledcontrols. The one or more controls may be used to measure an efficiencyof producing the labeled molecules.

The libraries disclosed herein may be used in a variety of applications.For example, the library could be used for sequencing applications. Thelibrary may be stored and used multiple times to generate samples foranalysis. Some applications include, for example, genotypingpolymorphisms, studying RNA processing, and selecting clonalrepresentatives to do sequencing.

Sample Preparation and Applications

The oligonucleotides (e.g., molecular bar code, sample tag, molecularlabel, cellular label) disclosed herein may be used in a variety ofmethods. The oligonucleotides may be in methods for nucleic acidanalysis. Nucleic acid analysis may include, but is not limited to,genotyping, gene expression, copy number variation, and molecularcounting.

The disclosure provides for methods of multiplex nucleic acid analysis.The method may comprise (a) contacting one or more oligonucleotides froma cell with one or more oligonucleotides attached to a support, whereinthe one or more oligonucleotides attached to the support comprise (i) acell label region comprising two or more randomer sequences connected bya non-random sequence; and (ii) a molecular label region; and (b)conducting one or more assays on the one or more oligonucleotides fromthe cell.

Further disclosed herein are methods of producing single cell nucleicacid libraries. The method may comprise (a) contacting one or moreoligonucleotides from a cell with one or more oligonucleotides attachedto a support, wherein the one or more oligonucleotides attached to thesupport comprise (i) a cell label region comprising two or more randomersequences connected by a non-random sequence; and (ii) a molecular labelregion; and (b) conducting one or more assays on the one or moreoligonucleotides from the cell.

In some instances, the method comprises adding a one or more cells ontoa microwell array. The number of cells to be added may be determinedfrom counting. Excess or unbound cells may be washed away using a buffer(e.g., phosphobuffered saline buffer, HEPES, Tris). The number of cellsthat may be captured by the wells of the microwell array may be relatedto the size of the cell. For example, depending on the design of themicrowell, larger cells may be more easily captured than smaller cells,as depicted in FIG. 6. Different microwells (e.g., different dimensions)may be used for capturing different cell types.

The methods described here allow for the addition of sequences that cannucleic acids for sequencing or other molecular analyses. These methodscan allow detection of nucleic acid variants, mutants, polymorphisms,inversions, deletions, reversions and other qualitative events found ina population of RNA or DNA molecules. For example, the methods can allowfor identification of target frequencies (e.g., gene expression orallelic distribution). For example, the methods also allow foridentification of mutations or SNPs in a genome or transcriptome, suchas from a diseased or non-diseased subject. The methods also allow fordetermining the presence or absence of contamination or infections in abiological sample from a subject, such as foreign organisms or viruses,such as a bacteria or a fungus.

Cells can be added into microwells by any method. In some embodiments,cells are added to microwells as a diluted cell sample. In someembodiments, cells are added to microwells and allowed to settle in themicrowells by gravity. In some embodiments, cells are added tomicrowells and centrifugation is used to settle the cells in themicrowells. In some embodiments, cells are added to microwells byinjecting one or more cells into one or more microwells. For example, asingle cell can be added to a microwell by injecting the single cell into a microwell. The injecting of a cell can be through the use of anydevice or method, such as through the use of a micro manipulator. Insome embodiments, cell can be added to microwells using a magnet. Forexample, cells can coated on their surface with magnetic particles, suchas magnetic microparticles or magnetic nanoparticles and added tomicrowells using a magnet or a magnetic field.

The microwell array comprising cells may be contacted with anoligonucleotide conjugated solid support (e.g., bead). Uncapturedoligonucleotide conjugated solid supports may be removed (e.g., washedaway with buffer). FIG. 5 depicts a microwell array with captured solidsupports. A microwell may comprise at least one solid support. Amicrowell may comprise at least two solid supports. A microwell maycomprise at most one solid support. A microwell may comprise at most twosolid supports. A microwell may comprise at least about 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more solid supports. A microwell may comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more solid supports. Someof the microwells of the microwell array may comprise one solid supportand some of the microwells of the microwell array may comprise two ormore solid supports, as shown in FIG. 5. The microwell may not need tobe covered for any of the methods of the disclosure. In other words,microwells may not need to be sealed during the method. When themicrowells are not covered (e.g., sealed), the wells may be spaced apartsuch that the contents of one microwell may not diffuse into anothermicrowell.

Alternatively, or additionally, cells may be captured and/or purifiedprior to being contacted with an oligonucleotide conjugated support.Methods to capture and/or purify cells may comprise use of antibodies,molecular scaffolds, and/or beads. Cells may be purified by flowcytometry. Commercially available kits may be used to capture or purifycells. For example, Dynabeads® may be used to isolate cells. Magneticisolation may be used to purify cells. Cells may be purified bycentrifugation.

Cells may be contacted with oligonucleotide conjugated supports bycreating a suspension comprising cells and the supports. The suspensionmay comprise a gel. Cells may be immobilized on a support or in asolution prior to contact with the oligonucleotide conjugated supports.Alternatively, cells may be added to a suspension comprising theoligonucleotide conjugated support. For example, cells may be added to ahydrogel that is embedded with oligonucleotide conjugated supports.

A single cell may be contacted with a single oligonucleotide coupledsolid support. A single cell may be contacted with multipleoligonucleotide conjugated solid supports. Multiple cells may interactwith a single oligonucleotide conjugated solid support. Multiple cellsmay interact with multiple oligonucleotide conjugated solid supports.The oligonucleotide conjugated solid supports may be cell-type specific.Alternatively, the oligonucleotide conjugated support may interact withtwo or more different cell types.

Lysis

Cells in the microwells may be lysed. Lysis may be performed bymechanical lysis, heat lysis, optical lysis, and/or chemical lysis.Chemical lysis may include the use of digestive enzymes such asproteinase K, pepsin, and trypsin. Lysis may be performed by theaddition of a lysis buffer to the microwells. A lysis buffer maycomprise Tris HCl. A lysis buffer may comprise at least about 0.01,0.05, 0.1, 0.5, or 1M or more Tris HCl. A lysis buffer may comprise atmost about 0.01, 0.05, 0.1, 0.5, or 1M or more Tris HCL. A lysis buffermay comprise about 0.1 M Tris HCl. The pH of the lysis buffer may be atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The pH of thelysis buffer may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore. In some instances, the pH of the lysis buffer is about 7.5. Thelysis buffer may comprise a salt (e.g., LiCl). The concentration of saltin the lysis buffer may be at least about 0.1, 0.5, or 1M or more. Theconcentration of salt in the lysis buffer may be at most about 0.1, 0.5,or 1M or more. In some instances, the concentration of salt in the lysisbuffer is about 0.5M. The lysis buffer may comprise a detergent (e.g.,SDS, Li dodecyl sufate, triton X, tween, NP-40). The concentration ofthe detergent in the lysis buffer may be at least about 0.0001, 0.0005,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or 7% or more. Theconcentration of the detergent in the lysis buffer may be at most about0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or7% or more. In some instances, the concentration of the detergent in thelysis buffer is about 1% Li dodecyl sulfate. The time used in the methodfor lysis may be dependent on the amount of detergent used. In someinstances, the more detergent used, the less time needed for lysis. Thelysis buffer may comprise a chelating agent (e.g., EDTA, EGTA). Theconcentration of a chelating agent in the lysis buffer may be at leastabout 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of achelating agent in the lysis buffer may be at most about 1, 5, 10, 15,20, 25, or 30 mM or more. In some instances, the concentration ofchelating agent in the lysis buffer is about 10 mM. The lysis buffer maycomprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). Theconcentration of the reducing reagent in the lysis buffer may be atleast about 1, 5, 10, 15, or 20 mM or more. The concentration of thereducing reagent in the lysis buffer may be at most about 1, 5, 10, 15,or 20 mM or more. In some instances, the concentration of reducingreagent in the lysis buffer is about 5 mM. In some instances, a lysisbuffer may comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl,about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

Lysis may be performed at a temperature of about 4, 10, 15, 20, 25, or30 C. Lysis may be performed for about 1, 5, 10, 15, or 20 or moreminutes. A lysed cell may comprise at least about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. A lysed cell may comprise at most about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. FIG. 7 illustrates exemplary statistics about theconcentration of target nucleic acid (i.e., mRNA) that may be obtainedfrom lysis.

Sealing

The microwells of the microwell array may be sealed during lysis.Sealing may be useful for preventing cross hybridization of targetnucleic acid between adjacent microwells. A microwell may be sealedusing a cap as shown in FIGS. 8A and B. A cap may be a solid support. Acap may comprise a bead. The diameter of the bead may be larger than thediameter of the microwell. For example, a cap may be at least about 10,20, 30, 40, 50, 60, 70, 80 or 90% larger than the diameter of themicrowell. For example, a cap may be at most about 10, 20, 30, 40, 50,60, 70, 80 or 90% larger than the diameter of the microwell.

A cap may comprise cross-linked dextran beads (e.g., Sephadex).Cross-linked dextran may range from about 10 micrometers to about 80micrometers. The cross-linked dextran of the cap may be from 20micrometers to about 50 micrometers. A cap may comprise, for example,anopore inorganic membranes (e.g., aluminum oxides), dialysis membranes,glass slides, coverslips, and/or hydrophilic plastic film (e.g., filmcoated with a thin film of agarose hydrated with lysis buffer).

The cap may allow buffer to pass through into and out of the microwell,but may prevent macromolecules (e.g., nucleic acid) from migrating outof the well. A macromolecule of at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides may beblocked from migrating into or out of the microwell by the cap. Amacromolecule of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13,14, 15, 16, 17, 18, 19, or 20 or more nucleotides may be blocked frommigrating into or out of the microwell by the cap.

A sealed microwell array may comprise a single layer of beads on top ofthe microwells. A sealed microwell array may comprise multiple layers ofbeads on top of the microwells. A sealed microwell array may compriseabout 1, 2, 3, 4, 5, or 6 or more layers of beads.

Depositing a bead, or plurality of beads, onto a solid support (e.g., amicrowell array) can be random or non-random. For example, contacting abead with a microwell array can be a random or non-random contacting. Insome embodiments, the bead is contacted with a microwell array randomly.In some embodiments, the bead is contacted with a microwell arraynon-randomly. Depositing of a plurality of beads to a microwell arraycan be random or non-random. For example, the contacting of a pluralityof beads to a microwell array can be a random or non-random contacting.In some embodiments, the plurality of beads is contacted to a microwellarray randomly. In some embodiments, the plurality of beads is contactedto a microwell array non-randomly.

Stochastic Labeling of Molecules

Wherein the sample tag or molecular identifier label is anoligonucleotide, attachment of the oligonucleotide to a nucleic acid mayoccur by a variety of methods, including, but not limited to,hybridization of the oligonucleotide to the nucleic acid. In someinstances, the oligonucleotide comprises a target specific region. Thetarget specific region may comprise a sequence that is complementary toat least a portion of the molecule to be labeled. The target specificregion may hybridize to the molecule, thereby producing a labelednucleic acid. Hybridization of the oligonucleotide to the nucleic acidmay be followed by a nucleic acid extension reaction. The nucleic acidextension reaction may be reverse transcription.

Attaching, alternatively referred to as contacting, the plurality ofnucleic acids with the sample tag may comprise hybridizing the sampletag to one or more of the plurality of nucleic acids. Contacting theplurality of nucleic acids with the sample tag may comprise performing anucleic acid extension reaction. The nucleic acid extension reaction maybe a reverse transcription reaction.

Contacting the plurality of nucleic acids with the molecular identifierlabel may comprise hybridizing the molecular identifier label to one ormore of the plurality of nucleic acids. Contacting the plurality ofnucleic acids with the molecular identifier label may compriseperforming a nucleic acid extension reaction. The nucleic acid extensionreaction may comprise reverse transcription.

Contacting the plurality of nucleic acids with the molecular identifierlabel may comprise hybridizing the sample tag to one or more of theplurality of nucleic acids. Contacting the plurality of nucleic acidswith the molecular identifier label may comprise hybridizing themolecular identifier label to the sample tag.

Contacting the plurality of nucleic acids with the sample tag maycomprise hybridizing the molecular identifier label to one or more ofthe plurality of nucleic acids. Contacting the plurality of nucleicacids with the sample tag may comprise hybridizing the sample tag to themolecular identifier label.

Attachment of the sample tag and/or the molecular identifier label to anucleic acid may occur by ligation. Contacting the plurality of nucleicacids with the sample tag may comprise ligating the sample tag to anyone of the plurality of nucleic acids. Contacting the plurality ofnucleic acids with the molecular identifier label may comprise ligatingthe molecular identifier label to one or more of the plurality ofnucleic acids. Contacting the plurality of nucleic acids with the sampletag may comprise ligating the molecular identifier label one or more thenucleic acids. Contacting the plurality of nucleic acids with themolecular identifier label may comprise ligating the sample tag to oneor more of the nucleic acids. Ligation techniques comprise blunt-endligation and sticky-end ligation. Ligation reactions may include DNAligases such as DNA ligase I, DNA ligase III, DNA ligase IV, and T4 DNAligase. Ligation reactions may include RNA ligases such as T4 RNA ligaseI and T4 RNA ligase II.

Methods of ligation are described, for example in Sambrook et al. (2001)and the New England BioLabs catalog both of which are incorporatedherein by reference for all purposes. Methods include using T4 DNALigase which catalyzes the formation of a phosphodiester bond betweenjuxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNAwith blunt and sticky ends; Taq DNA Ligase which catalyzes the formationof a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyltermini of two adjacent oligonucleotides which are hybridized to acomplementary target DNA; E. coli DNA ligase which catalyzes theformation of a phosphodiester bond between juxtaposed 5′-phosphate and3′-hydroxyl termini in duplex DNA containing cohesive ends; and T4 RNAligase which catalyzes ligation of a 5′ phosphoryl-terminated nucleicacid donor to a 3′ hydroxyl-terminated nucleic acid acceptor through theformation of a 3′.fwdarw.5′ phosphodiester bond, substrates includesingle-stranded RNA and DNA as well as dinucleoside pyrophosphates; orany other methods described in the art. Fragmented DNA may be treatedwith one or more enzymes, for example, an endonuclease, prior toligation of adaptors to one or both ends to facilitate ligation bygenerating ends that are compatible with ligation.

In some instances, both ends of the oligonucleotide are attached to themolecule. For example, both ends of the oligonucleotide may behybridized and/or ligated to one or more ends of the molecule. In someinstances, attachment of both ends of the oligonucleotide to both endsof the molecule results in the formation of a circularized labelednucleic acid. Both ends of the oligonucleotide may also be attached tothe same end of the molecule. For example, the 5′ end of theoligonucleotide is ligated to the 3′ end of the molecule and the 3′ endof the oligonucleotide is hybridized to the 3′ end of the molecule,resulting in a labeled nucleic acid with a hairpin structure at one end.In some instances the oligonucleotide is attached to the middle of themolecule.

In some instances, attachment of the oligonucleotide to the nucleic acidcomprises attaching one or more oligonucleotide linkers to the pluralityof nucleic acids. The method may further comprise attaching one or moreoligonucleotide linkers to the sample-tagged nucleic acids. The methodmay further comprise attaching one or more oligonucleotide linkers tothe labeled nucleic acids. Attaching one or more oligonucleotide linkersto a nucleic acid, sample tag or molecular identifier label may compriseligating one or more oligonucleotide linkers to a nucleic acid, sampletag or molecular identifier label. The one or more linkers may compriseat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100nucleotides. In some instances, the linker may comprise at least about1000 nucleotides.

In some instances, attachment of the molecular barcode to the moleculecomprises the use of one or more adaptors. As used herein, the terms“adaptors” and “adaptor regions” may be used interchangeably. Adaptorsmay comprise a target specific region, which allows the attachment ofthe adaptor to the molecule, and an oligonucleotide specific region,which allows attachment of the molecular barcode to the adaptor.Adaptors may further comprise a universal primer. Adaptors may furthercomprise a universal PCR region. Adaptors may be attached to themolecule and/or molecular barcodes by methods including, but not limitedto, hybridization and/or ligation.

Methods for ligating adaptors to fragments of nucleic acid are wellknown. Adaptors may be double-stranded, single-stranded or partiallysingle-stranded. In some aspects, adaptors are formed from twooligonucleotides that have a region of complementarity, for example,about 10 to 30, or about 15 to 40 bases of perfect complementarity; sothat when the two oligonucleotides are hybridized together they form adouble stranded region. Optionally, either or both of theoligonucleotides may have a region that is not complementary to theother oligonucleotide and forms a single stranded overhang at one orboth ends of the adaptor. Single-stranded overhangs may be about 1 toabout 8 bases, or about 2 to about 4. The overhang may be complementaryto the overhang created by cleavage with a restriction enzyme tofacilitate “sticky-end” ligation. Adaptors may include other features,such as primer binding sites and restriction sites. In some aspects therestriction site may be for a Type IIS restriction enzyme or anotherenzyme that cuts outside of its recognition sequence, such as EcoP151(see, Mucke et al. J Mol Biol 2001, 312(4):687-698 and U.S. Pat. No.5,710,000 which is incorporated herein by reference in its entirety).

In some instances, stochastically counting the number of copies of anucleic acid in a plurality of samples comprises detecting the adaptor,a complement of the adaptor, a reverse complement of the adaptor or aportion thereof to determine the number of different labeled nucleicacids. Detecting the adaptor, a complement of the adaptor, a reversecomplement of the adaptor or a portion thereof may comprise sequencingthe adaptor, a complement of the adaptor, a reverse complement of theadaptor or a portion thereof.

The molecular barcode may be attached to any region of a molecule. Forexample, the molecular barcode may be attached to the 5′ or 3′ end of apolynucleotide (e.g., DNA, RNA). For example, the target-specific regionof the molecular barcode comprises a sequence that is complementary to asequence in the 5′ region of the molecule. The target-specific region ofthe molecular barcode may also comprise a sequence that is complementaryto a sequence in the 3′ region of the molecule. In some instances, themolecular barcode is attached a region within a gene or gene product.For example, genomic DNA is fragmented and a sample tag or molecularidentifier label is attached to the fragmented DNA. In other instances,an RNA molecule is alternatively spliced and the molecular barcode isattached to the alternatively spliced variants. In another example, thepolynucleotide is digested and the molecular barcode is attached to thedigested polynucleotide. In another example, the target-specific regionof the molecular barcode comprises a sequence that is complementary to asequence within the molecule.

A molecular barcode, sample tag (e.g., sample index), cellular label, ormolecular identifier label (e.g., molecular label) comprising a hairpinmay act as a probe for a hybridization chain reaction (HCR), and, thus,may be referred to as an HCR probe. The HCR probe may comprise amolecular barcode comprising a hairpin structure. The HCR probe maycomprise a sample tag comprising a hairpin structure. The HCR probe maycomprise a molecular identifier label comprising a hairpin structure.Further disclosed herein is a stochastic label-based hybridization chainreaction (HCR) method comprising stochastically labeling one or morenucleic acid molecules with an HCR probe, wherein the HCR probecomprises a molecular barcode comprising a hairpin and the one or morenucleic acid molecules act as initiators for a hybridization chainreaction. Further disclosed herein is a stochastic label-basedhybridization chain reaction (HCR) method comprising stochasticallylabeling one or more nucleic acid molecules with an HCR probe, whereinthe HCR probe comprises a sample tag comprising a hairpin and the one ormore nucleic acid molecules act as initiators for a hybridization chainreaction. Further disclosed herein is a stochastic label-basedhybridization chain reaction (HCR) method comprising stochasticallylabeling one or more nucleic acid molecules with an HCR probe, whereinthe HCR probe comprises a molecular identifier label comprising ahairpin and the one or more nucleic acid molecules act as initiators fora hybridization chain reaction.

The HCR probe may comprise a hairpin with an overhang region. Theoverhang region of the hairpin may comprise a target specific region.The overhang region may comprise an oligodT sequence. The samplecomprising the one or more nucleic acid molecules may be treated withone or more restriction nucleases prior to stochastic labeling. Theoverhang region may comprise a restriction enzyme recognition sequence.The sample comprising the one or more nucleic acid molecules may becontacted with one or more adapters prior to stochastic labeling toproduce an adapter-nucleic acid molecule hybrid. The overhang region andthe stem may be complementary to the one or more adapters. The HCR probemay comprise a hairpin with a loop. The loop of the HCR probe maycomprise a label region and/or sample index region.

Hybridization of a first HCR probe to the nucleic acid molecules mayresult in the formation of a labeled nucleic acid, wherein the first HCRprobe is linearized to produce a first linearized HCR probe. The firstlinearized HCR probe of the labeled nucleic acid may act as an initiatorfor hybridization of a second HCR probe to the labeled nucleic acid toproduce a labeled nucleic acid with two linearized HCR probes. Thesecond linearized HCR probe may act as an initiator for anotherhybridization reaction. This process may be repeated multiple times toproduce a labeled nucleic acid with multiple linearized HCR probes. Thedetectable labels on the HCR probe may enable detection of the labelednucleic acid. The detectable labels may be any type of label (e.g.,fluorphore, chromophore, small molecule, nanoparticle, hapten, enzyme,antibody, magnet). The detectable labels may comprise fragments of asingle label. The detectable labels may generate a detectable signalwhen they are in close proximity. When the HCR probe is a hairpin, thedetectable labels may be too far away to produce a detectable signal.When the HCR probe is linearized and multiple linearized HCR probes arehybridized together, the detectable labels may be in close enoughproximity to generate a detectable signal. For example, a HCR probe maycomprise two pyrene moieties as detectable labels. Alternatively, thedetectable labels may be nanoparticles. The stochastic label-based HCRmethod may enable attachment of multiple hairpin HCR probes to a labelednucleic acid, which may result in signal amplification. Stochasticlabel-based HCR may increase the sensitivity of detection, analysisand/or quantification of the nucleic acid molecules. Stochasticlabel-based HCR may increase the accuracy of detection, analysis, and/orquantification of one or more nucleic acid molecules.

After lysis the target nucleic acid of the cells may hybridize to theoligonucleotide conjugated to the solid support. The target nucleic acidmay hybridize to the target binding region of the oligonucleotide. Thenucleic acid may hybridize to any region of the olignucleotide.

In some instances, not all oligonucleotides may bind a target nucleicacid. This is because in some instances, the number of oligonucleotidesis larger than the number of target nucleic acids. The number ofoligonucleotides conjugated to a solid support may be 1, 2, 3, 4, 5, 6,7, 8, 9, or 10-fold more than the number of target nucleic acids in acell. At least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of theoligonucleotides may be bound by a target nucleic acid. At most 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% of the oligonucleotides may be boundby a target nucleic acid. In some instances, at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more differenttarget nucleic acids may be captured by the oligonucleotides on a solidsupport. In some instances, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90 or 100 or more different target nucleic acidsmay be captured by the oligonucleotides on a solid support.

In some instances, at least about 40, 50, 60, 70, 80, 90, 95, 96, 97,98, 99, or 100% of the number of copies of a target nucleic acid arebound to oligonucleotides on a solid support. In some instances, at mostabout 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the numberof copies of a target nucleic acid are bound to oligonucleotides on asolid support.

Retrieval

After lysis, the solid supports may be retrieved. Retrieval of the solidsupports may be performed by using a magnet. Retrieval of the solidsupports may be performed by melting the microwell array and/orsonication. Retrieval of the solid supports may comprise centrifugation.Retrieval of the solid supports may comprise size exclusion. In someinstances, at least about 50, 60, 70, 80, 90, 95, or 100% of the solidsupports are recovered from the microwells. In some instances, at mostabout 50, 60, 70, 80, 90, 95, or 100% of the solid supports arerecovered from the microwells.

Reverse Transcription

The methods disclosed herein may further comprise reverse transcriptionof a labeled-RNA molecule to produce a labeled-cDNA molecule. In someinstances, at least a portion of the oligonucleotide acts as a primerfor the reverse transcription reaction. The oligodT portion of theoligonucleotide may act as a primer for first strand synthesis of thecDNA molecule.

In some instances the labeled cDNA molecule may be used as a moleculefor a new stochastic labeling reaction. The labeled cDNA may have afirst tag or set of tags from attachment to the RNA prior to reversetranscription and a second tag or set of tags attached to the cDNAmolecule. These multiple labeling reactions can, for example, be used todetermine the efficiency of events that occur between the attachment ofthe first and second tags, e.g., an optional amplification reaction orthe reverse transcription reaction.

In another example, an oligonucleotide is attached to the 5′ end of anRNA molecule to produce a labeled-RNA molecule. Reverse transcription ofthe labeled-RNA molecule may occur by the addition of a reversetranscription primer. In some instances, the reverse transcriptionprimer is an oligodT primer, random hexanucleotide primer, or atarget-specific oligonucleotide primer. Generally, oligodT primers are12-18 nucleotides in length (SEQ ID NO: 1) and bind to the endogenouspoly(A)+ tail at the 3′ end of mammalian mRNA. Random hexanucleotideprimers may bind to mRNA at a variety of complementary sites.Target-specific oligonucleotide primers typically selectively prime themRNA of interest.

In some instances, the method comprises repeatedly reverse transcribingthe labeled-RNA molecule to produce multiple labeled-cDNA molecules. Themethods disclosed herein may comprise conducting at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reversetranscription reactions. The method may comprise conducting at leastabout 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100reverse transcription reactions.

Nucleic acid synthesis (e.g., cDNA synthesis) may be performed on theretrieved solid supports. Nucleic acid synthesis may be performed in atube and/or on a rotor to keep the solid supports suspended. Theresulting synthesized nucleic acid may be used in subsequent nucleicacid amplification and/or sequencing technologies. Nucleic acidsynthesis may comprise generating cDNA copies on a RNA attached to theoligonucleotide on the solid support. Generating cDNA copies maycomprise using a reverse transcriptase (RT) or DNA polymerases having RTactivity. This may result in the production of single-stranded cDNAmolecules. After nucleic acid synthesis, unused oligonucleotides may beremoved from the solid support. Removal of the oligonucleotides mayoccur by exonuclease treatment (e.g., by ExoI).

In some embodiments, nucleic acids can be removed from the solid supportusing chemical cleavage. For example, a chemical group or a modifiedbase present in a nucleic acid can be used to facilitate its removalfrom a solid support. For example, an enzyme can be used to remove anucleic acid from a solid support. For example, a nucleic acid can beremoved from a solid support through a restriction endonucleasedigestion. For example, treatment of a nucleic acid containing a dUTP orddUTP with uracil-d-glycosylase (UDG) can be used to remove a nucleicacid from a solid support. For example, a nucleic acid can be removedfrom a solid support using an enzyme that performs nucleotide excision,such as a base excision repair enzyme, such as an apurinic/apyrimidinic(AP) endonuclease. In some embodiments, a nucleic acid can be removedfrom a solid support using a photocleavable group and light. In someembodiments, a cleavable linker can be used to remove a nucleic acidfrom the solid support. For example, the cleavable linker can compriseat least one of biotin/avidin, biotin/streptavidin, biotin/neutravidin,Ig-protein A, a photo-labile linker, acid or base labile linker group,or an aptamer.

In some embodiments, nucleic acids are not amplified. In someembodiments, nucleic acids are not amplified prior to sequencing thenucleic acids. In some embodiments, nucleic acids not attached to asolid support can be directly sequenced without prior amplification. Insome embodiments, nucleic acids can be directly sequenced withoutperforming amplification when attached to a solid support, for example,nucleic acids attached to a solid support can be directly sequencedwhile attached to the solid support. In some embodiments, a nucleic acidthat has been removed from a solid support can be directly sequenced.For example, a nucleic acid that has been removed from a solid supportcan be directly sequenced without performing amplification. Anysequencing platform conducive to sequencing without amplification can beused to perform the sequencing.

Amplification

After the nucleic acid has been synthesized (e.g., reverse transcribed),it may be amplified. Amplification may be performed in a multiplexmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. Amplification may add sequencing adaptors to the nucleicacid. Amplification may be performed by polymerase chain reaction (PCR).PCR may refer to a reaction for the in vitro amplification of specificDNA sequences by the simultaneous primer extension of complementarystrands of DNA. PCR may encompass derivative forms of the reaction,including but not limited to, RT-PCR, real-time PCR, nested PCR,quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

The method may further comprise conducting one or more amplificationreactions to produce labeled nucleic acid amplicons. The labeled nucleicacids may be amplified prior to detecting the labeled nucleic acids. Themethod may further comprise combining the first and second samples priorto conducting the one or more amplification reactions.

The amplification reactions may comprise amplifying at least a portionof the sample tag. The amplification reactions may comprise amplifyingat least a portion of the label. The amplification reactions maycomprise amplifying at least a portion of the sample tag, label, nucleicacid, or a combination thereof. The amplification reactions may compriseamplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, or 100% of the plurality of nucleic acids. The method mayfurther comprise conducting one or more cDNA synthesis reactions toproduce one or more cDNA copies of the sample-tagged nucleic acids ormolecular identifier labeled nucleic acids.

Amplification of the labeled nucleic acids may comprise PCR-basedmethods or non-PCR based methods. Amplification of the labeled nucleicacids may comprise exponential amplification of the labeled nucleicacids. Amplification of the labeled nucleic acids may comprise linearamplification of the labeled nucleic acids.

In some instances, amplification of the labeled nucleic acids comprisesnon-PCR based methods. Examples of non-PCR based methods include, butare not limited to, multiple displacement amplification (MDA),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), strand displacement amplification (SDA),real-time SDA, rolling circle amplification, or circle-to-circleamplification. Other non-PCR-based amplification methods includemultiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptionamplification or RNA-directed DNA synthesis and transcription to amplifyDNA or RNA targets (WO 89/01050; WO 88/10315; and U.S. Pat. Nos.5,130,238; 5,409,818; 5,466,586; 5,514,545; 5,554,517; 5,888,779;6,063,603; and 6,197,554), a ligase chain reaction (LCR), a Qβ replicase(Qβ) method as described in U.S. Pat. No. 4,786,600, use of palindromicprobes, strand displacement amplification, oligonucleotide-drivenamplification using a restriction endonuclease, an amplification methodin which a primer is hybridized to a nucleic acid sequence and theresulting duplex is cleaved prior to the extension reaction andamplification, strand displacement amplification using a nucleic acidpolymerase lacking 5′ exonuclease activity (U.S. Pat. No. 6,214,587),rolling circle amplification, and ramification extension amplification(RAM) (U.S. Pat. No. 5,942,391).

Amplification of the labeled nucleic acids may comprise hybridizationchain reaction (HCR) based methods (Dirks and Pierce, PNAS, 2004; Zhanget al., Anal Chem, 2012). HCR based methods may comprise DNA-based HCR.HCR based methods may comprise one or more labeled probes. The one ormore labeled probes may comprise one or more sample tags or molecularidentifier labels, or the complement thereof, disclosed herein.

In some instances, the methods disclosed herein further compriseconducting a polymerase chain reaction on the labeled nucleic acid(e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce alabeled-amplicon. The labeled-amplicon may be double-stranded molecule.The double-stranded molecule may comprise a double-stranded RNAmolecule, a double-stranded DNA molecule, or a RNA molecule hybridizedto a DNA molecule. One or both of the strands of the double-strandedmolecule may comprise the sample tag or molecular identifier label.Alternatively, the labeled-amplicon is a single-stranded molecule. Thesingle-stranded molecule may comprise DNA, RNA, or a combinationthereof. The nucleic acids of the present invention may comprisesynthetic or altered nucleic acids.

The polymerase chain reaction may be performed by methods such as PCR,HD-PCR, Next Gen PCR, digital RTA, or any combination thereof.Additional PCR methods include, but are not limited to, allele-specificPCR, Alu PCR, assembly PCR, asymmetric PCR, droplet PCR, emulsion PCR,helicase dependent amplification HDA, hot start PCR, inverse PCR,linear-after-the-exponential (LATE)-PCR, long PCR, multiplex PCR, nestedPCR, hemi-nested PCR, quantitative PCR, RT-PCR, real time PCR, singlecell PCR, touchdown PCR or combinations thereof.

Multiplex PCR reactions may comprise nested PCR reactions. The methodmay comprise a pair of primers wherein a first primer that anneals toany one of the plurality of nucleic acids at least 300 to 400nucleotides from the 3′ end of any one of the plurality of nucleic acidsand a second primer that anneals to any one of the plurality of nucleicacids at least 200 to 300 nucleotides from the 3′ end of any one of theplurality of nucleic acids, wherein the first primer and second primergenerate complementary DNA synthesis towards the 3′ end of any one ofthe plurality of nucleic acids.

In some instances, conducting a polymerase chain reaction comprisesannealing a first target specific primer to the labeled nucleic acid.Alternatively or additionally, conducting a polymerase chain reactionfurther comprises annealing a universal primer to a universal primerbinding site region of the sample tag or molecular identifier label,wherein the sample tag or molecular identifier label is on a labelednucleic acid or labeled-amplicon. The methods disclosed herein mayfurther comprise annealing a second target specific primer to thelabeled nucleic acid and/or labeled-amplicon.

In some instances, the method comprises repeatedly amplifying thelabeled nucleic acid to produce multiple labeled-amplicons. The methodsdisclosed herein may comprise conducting at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplificationreactions. Alternatively, the method comprises conducting at least about25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100amplification reactions.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909,5,861,245), rolling circle amplification (RCA) (for example, Fire andXu, PNAS 92:4641 (1995) and Liu et al., J. Am. Chem. Soc. 118:1587(1996)) and U.S. Pat. No. 5,648,245, strand displacement amplification(see Lasken and Egholm, Trends Biotechnol. 2003 21(12):531-5; Barker etal. Genome Res. 2004 May; 14(5):901-7; Dean et al. Proc Natl Acad SciUSA 2002; 99(8):5261-6; Walker et al. 1992, Nucleic Acids Res.20(7):1691-6, 1992 and Paez, et al. Nucleic Acids Res. 2004; 32(9):e71),Qbeta Replicase, described in PCT Patent Application No. PCT/US87/00880and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference), Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 6,582,938, 5,242,794, 5,494,810, 4,988,617,and US Pub. No. 20030143599 each of which is incorporated herein byreference. DNA may also be amplified by multiplex locus-specific PCR orusing adaptor-ligation and single primer PCR (See Kinzler andVogelstein, NAR (1989) 17:3645-53. Other available methods ofamplification, such as balanced PCR (Makrigiorgos, et al. (2002), NatBiotechnol, Vol. 20, pp. 936-9), may also be used.

Molecular inversion probes (“MIPs”) may also be used for amplificationof selected targets. MIPs may be generated so that the ends of thepre-circle probe are complementary to regions that flank the region tobe amplified. The gap may be closed by extension of the end of the probeso that the complement of the target is incorporated into the MIP priorto ligation of the ends to form a closed circle. The closed circle maybe amplified and detected by sequencing or hybridization as previouslydisclosed in Hardenbol et al., Genome Res. 15:269-275 (2005) and in U.S.Pat. No. 6,858,412.

Amplification may further comprise adding one or more control nucleicacids to one or more samples comprising a plurality of nucleic acids.Amplification may further comprise adding one or more control nucleicacids to a plurality of nucleic acids. The control nucleic acids maycomprise a control label.

Amplification may comprise use of one or more non-natural nucleotides.Non-natural nucleotides may comprise photolabile and/or triggerablenucleotides. Examples of non-natural nucleotides include, but are notlimited to, peptide nucleic acid (PNA), morpholino and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Non-natural nucleotides may be added to one or more cyclesof an amplification reaction. The addition of the non-naturalnucleotides may be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions may comprise the useof one or more primers. The one or more primers may comprise one or moreoligonucleotides. The one or more oligonucleotides may comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides may compriseless than 12-15 nucleotides. The one or more primers may anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers may anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers may anneal to an internalregion of the plurality of labeled nucleic acids. The internal regionmay be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theone or more primers may comprise a fixed panel of primers. The one ormore primers may comprise at least one or more custom primers. The oneor more primers may comprise at least one or more control primers. Theone or more primers may comprise at least one or more housekeeping geneprimers. The one or more oligonucleotides may comprise a sequenceselected from a group consisting of sequences in Table 23. The one ormore primers may comprise a universal primer. The universal primer mayanneal to a universal primer binding site. The one or more customprimers may anneal to the first sample tag, the second sample tag, themolecular identifier label, the nucleic acid or a product thereof. Theone or more primers may comprise a universal primer and a custom primer.The custom primer may be designed to amplify one or more target nucleicacids. The target nucleic acids may comprise a subset of the totalnucleic acids in one or more samples. The target nucleic acids maycomprise a subset of the total labeled nucleic acids in one or moresamples. The one or more primers may comprise at least 96 or more customprimers. The one or more primers may comprise at least 960 or morecustom primers. The one or more primers may comprise at least 9600 ormore custom primers. The one or more custom primers may anneal to two ormore different labeled nucleic acids. The two or more different labelednucleic acids may correspond to one or more genes.

Disclosed herein is a method of selecting a custom primer comprising: a)a first pass, wherein primers chosen may comprise: i) no more than threesequential guanines, no more than three sequential cytosines, no morethan four sequential adenines, and no more than four sequentialthymines; ii) at least 3, 4, 5, or 6 nucleotides that are guanines orcytosines; and iii) a sequence that does not easily form a hairpinstructure; b) a second pass, comprising: i) a first round of choosing aplurality of sequences that have high coverage of all transcripts; andii) one or more subsequent rounds, selecting a sequence that has thehighest coverage of remaining transcripts and a complementary score withother chosen sequences no more than 4; and c) adding sequences to apicked set until coverage saturates or total number of customer primersis less than or equal to about 96.

The method of selecting the custom primer may further comprise selectingthe at least one common primer based on one or more mRNA transcripts,non-coding transcripts including structural RNAs, transcribedpseudogenes, model mRNA provided by a genome annotation process,sequences corresponding to the genomic contig, or any combinationthereof.

The method of selecting the custom primer may further comprise a primerselection method that enriches for one or more subsets of nucleic acids.The one or more subsets may comprise low abundance mRNAs.

The method of selecting the custom primer may further comprise acomputational algorithm. Primers used in the method may be designed withthe use of the Primer 3, a computer program which suggests primersequences based on a user defined input sequence. Other primer designsmay also be used, or primers may be selected by eye without the aid ofcomputer programs. There are many options available with the program totailor the primer design to most applications. Primer3 may consider manyfactors, including, but not limited to, oligo melting temperature,length, GC content, 3′ stability, estimated secondary structure, thelikelihood of annealing to or amplifying undesirable sequences (forexample interspersed repeats) and the likelihood of primer-dimerformation between two copies of the same primer. In the design of primerpairs, Primer3 may consider product size and melting temperature, thelikelihood of primer-dimer formation between the two primers in thepair, the difference between primer melting temperatures, and primerlocation relative to particular regions of interest to be avoided.

The methods, compositions and kits disclosed herein may comprise one ormore primers disclosed in Tables 23-24.

Sequencing

In some aspects, determining the number of different labeled nucleicacids may comprise determining the sequence of the labeled nucleic acidor any product thereof (e.g., labeled-amplicons, labeled-cDNAmolecules). In some instances, an amplified target nucleic acid may besubjected to sequencing. Determining the sequence of the labeled nucleicacid or any product thereof may comprise conducting a sequencingreaction to determine the sequence of at least a portion of the sampletag, molecular identifier label, at least a portion of the labelednucleic acid, a complement thereof, a reverse complement thereof, or anycombination thereof. In some instances only the sample tag or a portionof the sample tag is sequenced. In some instances only the molecularidentifier label or a portion of the molecular identifier label issequenced.

Determining the sequence of the labeled nucleic acid or any productthereof may be performed by sequencing methods such as Helioscope™single molecule sequencing, Nanopore DNA sequencing, Lynx Therapeutics'Massively Parallel Signature Sequencing (MPSS), 454 pyrosequencing,Single Molecule real time (RNAP) sequencing, Illumina (Solexa)sequencing, SOLiD sequencing, Ion Torrent™, Ion semiconductorsequencing, Single Molecule SMRT™ sequencing, Polony sequencing, DNAnanoball sequencing, and VisiGen Biotechnologies approach.Alternatively, determining the sequence of the labeled nucleic acid orany product thereof may use sequencing platforms, including, but notlimited to, Genome Analyzer IIx, HiSeq, and MiSeq offered by Illumina,Single Molecule Real Time (SMRT™) technology, such as the PacBio RSsystem offered by Pacific Biosciences (California) and the SolexaSequencer, True Single Molecule Sequencing (tSMS™) technology such asthe HeliScope™ Sequencer offered by Helicos Inc. (Cambridge, Mass.).

In some embodiments, the labeled nucleic acids comprise nucleic acidsrepresenting from about 0.01% of the genes of an organism's genome toabout 100% of the genes of an organism's genome. For example, about0.01% of the genes of an organism's genome to about 100% of the genes ofan organism's genome can be sequenced using a target complimentaryregion comprising a plurality of multimers by capturing the genescontaining a complimentary sequence from the sample. In someembodiments, the labeled nucleic acids comprise nucleic acidsrepresenting from about 0.01% of the transcripts of an organism'stranscriptome to about 100% of the transcripts of an organism'stranscriptome. For example, about 0.501% of the transcripts of anorganism's transcriptome to about 100% of the transcripts of anorganism's transcriptome can be sequenced using a target complimentaryregion comprising a poly-T tail by capturing the mRNAs from the sample.

In some instances, determining the sequence of the labeled nucleic acidor any product thereof comprises paired-end sequencing, nanoporesequencing, high-throughput sequencing, shotgun sequencing,dye-terminator sequencing, multiple-primer DNA sequencing, primerwalking, Sanger dideoxy sequencing, Maxim-Gilbert sequencing,pyrosequencing, true single molecule sequencing, or any combinationthereof. Alternatively, the sequence of the labeled nucleic acid or anyproduct thereof may be determined by electron microscopy or achemical-sensitive field effect transistor (chemFET) array.

Determination of the sequence of a nucleic acid (e.g., amplified nucleicacid, labeled nucleic acid, cDNA copy of a labeled nucleic acid, etc.)may be performed using variety of sequencing methods including, but notlimited to, sequencing by hybridization (SBH), sequencing by ligation(SBL), quantitative incremental fluorescent nucleotide additionsequencing (QIFNAS), stepwise ligation and cleavage, fluorescenceresonance energy transfer (FRET), molecular beacons, TaqMan reporterprobe digestion, pyrosequencing, fluorescent in situ sequencing(FISSEQ), FISSEQ beads, wobble sequencing, multiplex sequencing,polymerized colony (POLONY) sequencing; nanogrid rolling circlesequencing (ROLONY), allele-specific oligo ligation assays (e.g., oligoligation assay (OLA), single template molecule OLA using a ligatedlinear probe and a rolling circle amplification (RCA) readout, ligatedpadlock probes, and/or single template molecule OLA using a ligatedcircular padlock probe and a rolling circle amplification (RCA) readout)and the like. High-throughput sequencing methods, such as cyclic arraysequencing using platforms such as Roche 454, Illumina Solexa,ABI-SOLiD, ION Torrents, Complete Genomics, Pacific Bioscience, Helicos,Polonator platforms, may also be utilized. Sequencing may comprise MiSeqsequencing. Sequencing may comprise HiSeq sequencing. Sequencing mayread the cell label, the molecular label and/or the gene that was on theoriginal oligonucleotide.

In another example, determining the sequence of labeled nucleic acids orany product thereof comprises RNA-Seq or microRNA sequencing.Alternatively, determining the sequence of labeled nucleic acids or anyproducts thereof comprises protein sequencing techniques such as Edmandegradation, peptide mass fingerprinting, mass spectrometry, or proteasedigestion.

The sequencing reaction can, in certain embodiments, occur on a solid orsemi-solid support, in a gel, in an emulsion, on a surface, on a bead,in a drop, in a continuous follow, in a dilution, or in one or morephysically separate volumes.

Sequencing may comprise sequencing at least about 10, 20, 30, 40, 50,60, 70, 80, 90, 100 or more nucleotides or base pairs of the labelednucleic acid. In some instances, sequencing comprises sequencing atleast about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or morenucleotides or base pairs of the labeled nucleic acid. In otherinstances, sequencing comprises sequencing at least about 1500; 2,000;3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or morenucleotides or base pairs of the labeled nucleic acid.

Sequencing may comprise at least about 200, 300, 400, 500, 600, 700,800, 900, 1000 or more sequencing reads per run. In some instances,sequencing comprises sequencing at least about 1500; 2,000; 3,000;4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 or more sequencingreads per run. Sequencing may comprise less than or equal to about1,600,000,000 sequencing reads per run. Sequencing may comprise lessthan or equal to about 200,000,000 reads per run.

Determining the number of different labeled nucleic acids may compriseone or more arrays.

Determining the number of different labeled nucleic acids may comprisecontacting the labeled nucleic acids with the one or more probes.

Probes, as described herein, may comprise a sequence that iscomplementary to at least a portion of the labeled nucleic acid orlabeled-amplicon. The plurality of probes may be arranged on the solidsupport in discrete regions, wherein a discrete region on the solidsupport comprises probes of identical or near-identical sequences. Insome instances, two or more discrete regions on the solid supportcomprise two different probes comprising sequences complementary to thesequence of two different unique identifier regions of theoligonucleotide tag.

In some instances, the plurality of probes is hybridized to the array.The plurality of probes may allow hybridization of the labeled-moleculeto the array. The plurality of probes may comprise a sequence that iscomplementary to the stochastic label oligo dT. Alternatively, oradditionally, the plurality of probes comprises a sequence that iscomplementary to the molecule.

Determining the number of different labeled nucleic acids may comprisecontacting the labeled nucleic acids with an array of a plurality ofprobes. Determining the number of different labeled nucleic acids maycomprise contacting the labeled nucleic acids with a glass slide of aplurality of probes.

Determining the number of different labeled nucleic acids may compriselabeled probe hybridization, target-specific amplification,target-specific sequencing, sequencing with labeled nucleotides specificfor target small nucleotide polymorphism, sequencing with labelednucleotides specific for restriction enzyme digest patterns, sequencingwith labeled nucleotides specific for mutations, or a combinationthereof.

Determining the number of different labeled nucleic acids may compriseflow cytometry sorting of a sequence-specific label. Determining thenumber of different labeled nucleic acids may comprise detection of thelabeled nucleic acids attached to the beads. Detection of the labelednucleic acids attached to the beads may comprise fluorescence detection.

Determining the number of different labeled nucleic acids may comprisecounting the plurality of labeled nucleic acids by fluorescenceresonance energy transfer (FRET), between a target-specific probe and alabeled nucleic acid or a target-specific labeled probe.

Detection of Labeled Nucleic Acids

The methods disclosed herein may further comprise detection of thelabeled nucleic acids and/or labeled-amplicons. Detection of the labelednucleic acids and/or labeled-amplicons may comprise hybridization of thelabeled nucleic acids to surface, e.g., a solid support. The method mayfurther comprise immunoprecipitation of a target sequence with anucleic-acid binding protein. Detection of the labeled nucleic acidsand/or labeled amplicons may enable or assist in determining the numberof different labeled nucleic acids.

In some instances, the method further comprises contacting the labelednucleic acids and/or labeled-amplicons with a detectable label toproduce a detectable-label conjugated labeled nucleic acid. The methodsdisclosed herein may further comprise detecting the detectable-labelconjugated labeled nucleic acid. Detection of the labeled nucleic acidsor any products thereof (e.g., labeled-amplicons, detectable-labelconjugated labeled nucleic acid) may comprise detection of at least aportion of the sample tag or molecular identifier label, molecule,detectable label, a complement of the sample tag or molecular identifierlabel, a complement of the molecule, or any combination thereof.

Detection of the labeled nucleic acids or any products thereof maycomprise an emulsion or a droplet. For example, the labeled nucleicacids or any products thereof may be in an emulsion or droplet. Adroplet can be a small volume of a first liquid that is encapsulated byan immiscible second liquid, such as a continuous phase of an emulsion(and/or by a larger droplet). The volume of a droplet, and/or theaverage volume of droplets in an emulsion, can, for example, be lessthan about one microliter (or between about one microliter and onenanoliter or between about one microliter and one picoliter), less thanabout one nanoliter (or between about one nanoliter and one picoliter),or less than about one picoliter (or between about one picoliter and onefemtoliter), among others. A droplet (or droplets of an emulsion) canhave a diameter (or an average diameter) of less than about 1000, 100,or 10 micrometers, or about 1000 to 10 micrometers, among others. Adroplet can be spherical or nonspherical. Droplets can be generatedhaving an average diameter of about, less than about, or more than about0.001, 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120,130, 140, 150, 160, 180, 200, 300, 400, or 500 microns. Droplets canhave an average diameter of about 0.001 to about 500, about 0.01 toabout 500, about 0.1 to about 500, about 0.1 to about 100, about 0.01 toabout 100, or about 1 to about 100 microns. A droplet can be a simpledroplet or a compound droplet. The term emulsion, as used herein, canrefer to a mixture of immiscible liquids (such as oil and water).Oil-phase and/or water-in-oil emulsions allow for thecompartmentalization of reaction mixtures within aqueous droplets. Theemulsions can comprise aqueous droplets within a continuous oil phase.The emulsions provided herein can be oil-in-water emulsions, wherein thedroplets are oil droplets within a continuous aqueous phase. When anemulsion or droplet is used to isolate, for example, spatially isolate,single cells, a solid support may not be used. Thus the nucleic acids tobe tagged and analyzed may not be bound to a solid support and in suchinstances; a cellular label can correspond to the single cell orpopulation of cells present in the emulsion or droplet when tagged. Theemulsion or droplet can thus effectively isolate the tagging or labelingsteps with a single cell or plurality of cells and the cellular labelcan be used to identify the nucleic acids that came from the single cellor plurality of cells. In some embodiments, droplets can be applied tomicrowells, for example, similarly to application of beads to microwellarrays.

Alternatively, detection of the labeled nucleic acids or any productsthereof comprises one or more solutions. In other instances, detectionof the labeled nucleic acids comprises one or more containers.

Detection of the labeled nucleic acids or any products thereof (e.g.,labeled-amplicons, detectable-label conjugated labeled nucleic acid) maycomprise detecting each labeled nucleic acid or products thereof. Forexample, the methods disclosed herein comprise sequencing at least aportion of each labeled nucleic acid, thereby detecting each labelednucleic acid.

In some instances, detection of the labeled nucleic acids and/orlabeled-amplicons comprises electrophoresis, spectroscopy, microscopy,chemiluminescence, luminescence, fluorescence, immunofluorescence,colorimetry, or electrochemiluminescence methods. For example, themethod comprises detection of a fluorescent dye. Detection of thelabeled nucleic acid or any products thereof may comprise colorimetricmethods. For example, the colorimetric method comprises the use of acolorimeter or a colorimetric reader. A non-limiting list ofcolorimeters and colorimetric readers include Sensovation's ColorimetricArray Imaging Reader (CLAIR), ESEQuant Lateral Flow Immunoassay Reader,SpectraMax 340PC 38, SpectraMax Plus 384, SpectraMax 190, VersaMax,VMax, and EMax.

Additional methods used alone or in combination with other methods todetect the labeled nucleic acids and/or amplicons may comprise the useof an array detector, fluorescence reader, non-fluorescent detector, CRreader, luminometer, or scanner. In some instances, detecting thelabeled nucleic acids and/or labeled-amplicons comprises the use of anarray detector. Examples of array detectors include, but are not limitedto, diode-array detectors, photodiode array detectors, HLPC photodiodearray detectors, array detectors, Germanium array detectors, CMOS andCCD array detectors, Gated linear CCD array detectors, InGaAs photodiodearray systems, and TE cooled CCD systems. The array detector may be amicroarray detector. Non-limiting examples of microarray detectorsinclude microelectrode array detectors, optical DNA microarray detectionplatforms, DNA microarray detectors, RNA microarray detectors, andprotein microarray detectors.

In some instances, a fluorescence reader is used to detect the labelednucleic acid and/or labeled-amplicons. The fluorescence reader may read1, 2, 3, 4, 5, or more color fluorescence microarrays or otherstructures on biochips, on slides, or in microplates. In some instances,the fluorescence reader is a Sensovation Fluorescence Array imagingReader (FLAIR). Alternatively, the fluorescence reader is a fluorescencemicroplate reader such as the Gemini XPS Fluorescence microplate reader,Gemini EM Fluorescence microplate reader, Finstruments® Fluoroskanfilter based fluorescence microplate reader, PHERAstar microplatereader, FlUOstar microplate reader, POLARstar Omega microplate reader,FLUOstar OPTIMA multi-mode microplate reader and POLARstar OPTIMAmulti-mode microplate reader. Additional examples of fluorescencereaders include PharosFX™ and PharosFX Plus systems.

In some instances, detection of the labeled nucleic acid and/orlabeled-amplicon comprises the use of a microplate reader. In someinstances, the microplate reader is an xMark™ microplate absorbancespectrophotometer, iMark microplate absorbance reader, EnSpire®Multimode plate reader, EnVision Multilabel plate reader, VICTOR XMultilabel plate reader, FlexStation, SpectraMax Paradigm, SpectraMaxM5e, SpectraMax M5, SpectraMax M4, SpectraMax M3, SpectraMax M2-M2e,FilterMax F series, Fluoroskan Ascent FL Microplate Fluoremeter andLuminometer, Fluoroskan Ascent Microplate Fluoremeter, Luminoskan AscentMicroplate Luminometer, Multiskan EX Microplate Photometer, Muliskan FCMicroplate Photometer, and Muliskan GO Microplate Photometer. In someinstances, the microplate reader detects absorbance, fluorescence,luminescence, time-resolved fluorescence, light scattering, or anycombination thereof. In some embodiments, the microplate reader detectsdynamic light scattering. The microplate reader, may in some instances,detect static light scattering. In some instances, detection of thelabeled nucleic acids and/or labeled-amplicons comprises the use of amicroplate imager. In some instances, the microplate imager comprisesViewLux uHTS microplate imager and BioRad microplate imaging system.

Detection of labeled nucleic acids and/or products thereof may comprisethe use of a luminometer. Examples of luminometers include, but are notlimited to, SpectraMax L, GloMax0-96 microplate luminometer,GloMax®-20/20 single-tube luminometer, GloMax®-Multi+ with Instinct™software, GloMax®-Multi Jr single tube multimode reader, LUMIstarOPTIMA, LEADER HC+ luminometer, LEADER 450i luminometer, and LEADER 50iluminometer.

In some instances, detection of the labeled nucleic acids and/orlabeled-amplicons comprises the use of a scanner. Scanners includeflatbed scanners such as those provided by Cannon, Epson, HP, Fujitsu,and Xerox. Additional examples of flatbed scanners include the FMBIO®fluorescence imaging scanners (e.g., FMBIO® II, III, and III Plussystems). Scanners may include microplate scanners such as the ArrayitArrayPix™ microarray microplate scanner. In some instances, the scanneris a Personal Molecular Imager™ (PMI) system provided by Bio-rad.

Detection of the labeled nucleic acid may comprise the use of ananalytical technique that measures the mass-to-charge ratio of chargedparticles, e.g., mass spectrometry. In some embodiments themass-to-charge ratio of charged particles is measured in combinationwith chromatographic separation techniques. In some embodimentssequencing reactions are used in combination with mass-to-charge ratioof charged particle measurements. In some embodiments the tags compriseisotopes. In some embodiments the isotope type or ratio is controlled ormanipulated in the tag library.

Detection of the labeled nucleic acids or any products thereof comprisesthe use of small particles and/or light scattering. For example, theamplified molecules (e.g., labeled-amplicons) are attached to haptens ordirectly to small particles and hybridized to the array. The smallparticles may be in the nanometer to micrometer range in size. Theparticles may be detected when light is scattered off of its surface.

A colorimetric assay may be used where the small particles are colored,or haptens may be stained with colorimetric detection systems. In someinstances, a flatbed scanner may be used to detect the light scatteredfrom particles, or the development of colored materials. The methodsdisclosed herein may further comprise the use of a light absorbingmaterial. The light absorbing material may be used to block undesirablelight scatter or reflection. The light absorbing material may be a foodcoloring or other material. In some instances, detection of the labelednucleic acid or any products thereof comprises contacting the labelednucleic acids with an off-axis white light.

In some embodiments, two or more different types of biological materialsfrom a sample can be detected simultaneously. For example, two or moredifferent types of biological materials selected from the groupconsisting of DNA, RNA (e.g., microRNA, mRNA, etc.), nucleotide,protein, and carbohydrate, from a sample can be detected simultaneously.For example, DNA and RNA from a sample can be detected simultaneouslyusing the methods described herein.

Data Analysis

The sequencing data may be used to count the number of target nucleicacid molecules in a cell. For example, a plurality of copies of a targetnucleic acid in a cell may bind to a different oligonucleotide on thesolid support. When the plurality of target nucleic acids are amplifiedand sequenced, they may comprise different molecular labels. The numberof molecular labels for a same target nucleic acid may be indicative ofthe number of copies of the target nucleic acid in the cell. Determiningthe copy number of a target nucleic acid may be useful for removingamplification bias when determining the concentration of a targetnucleic acid in a cell.

The sequencing data may be used to genotype a subject. By comparingtarget nucleic acids with different cellular labels, the copy numbervariation and/or concentration of the target nucleic acid may bedetermined. By comparing concentrations of target nucleic acids withdifferent cellular labels, the sequencing data may be used to determinecellular genotype heterogeneity. For example, a first cell of a samplemay comprise a target nucleic acid at high concentrations, whereas asecond cell of the sample may not comprise the target nucleic acid, ormay comprise the target nucleic acid at low concentrations, therebyindicating the heterogeneity of the cellular sample.

Determining cellular genotype heterogeneity may be useful fordiagnosing, prognosing, and determining a course of treatment of adisease. For example, if a first cell of a sample comprises the targetnucleic acid, but a second cell of the sample does not comprise thetarget nucleic acid, but comprises a second target nucleic acid, then acourse of a treatment may include an agent (e.g., drug) to target thefirst genotype and an agent (e.g., drug) to target the second genotype.

In some embodiments, certain sequence types can be linked to a DNA orRNA profile. For example, T-cell receptor and/or B-cell receptorsequences can be linked to a transcription profile, microRNA profile, orgenomic mutation profile of a sample, such as a single cell. In someembodiments, certain sequence types can be linked to an antigenicity orprotein expression profile. For example, T-cell receptor and/or B-cellreceptor sequences can be linked to to an antigenicity or proteinexpression profile via binding antibodies to a surface, such as asurface comprising proteins, such as protein targets of antibodiescomprising the T-cell receptor and/or B-cell receptor sequences.

In some embodiments, the presence or absence of a sequence, such as aviral sequence, can be linked to a DNA or RNA profile. For example, thepresence or absence of a sequence, such as a viral sequence, can belinked to a transcription profile, microRNA profile, or genomic mutationprofile of a sample, such as a single cell.

Kits

The present disclosure provides kits for carrying out the methods of thedisclosure. A kit may comprise one or more of: a microwell array, anoligonucleotide, and a solid support. A kit may comprise a reagent forreconstituting and/or diluting the oligonucleotides and/or solidsupport. A kit may comprise reagents for conjugating theoligonucleotides to the solid support. A kit may further comprise one ormore additional reagents, where such additional reagents may be selectedfrom: a wash buffer; a control reagent, an amplification agent foramplifying (e.g., performing cDNA synthesis and PCR) a target nucleicacid, and a conjugation agent for conjugating an oligonucleotide to thesolid support. Components of a subject kit may be in separatecontainers, or may be combined in a single container.

A kit may comprise instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods may be recorded on a suitable recording medium. Forexample, the instructions may be printed on a substrate, such as paperor plastic, etc. As such, the instructions may be present in the kits asa package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In some embodiments, the instructions may be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g., CD-ROM, diskette, flash drive, etc. In someembodiments, the actual instructions may not be present in the kit, butmeans for obtaining the instructions from a remote source, e.g., via theinternet, are provided. For example a kit may comprise a web addresswhere the instructions may be viewed and/or from which the instructionsmay be downloaded. As with the instructions, this means for obtainingthe instructions is recorded on a suitable substrate.

Further disclosed herein are kits for use in analyzing two or moremolecules from two or more samples. The kits disclosed herein maycomprise a plurality of beads, a primer and amplification agentssufficient to process at least about 384 samples. Any one of the samplesmay comprise a single cell. The nucleic acid amplification may result ina measurement of about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900 or 1000 targeted nucleic acids in a sample. The nucleic acidamplification may result in a measurement of about 1000 targeted nucleicacids in a sample. The nucleic acid amplification may result in ameasurement of about 100 targeted nucleic acids in a sample. The nucleicacid amplification may result in a measurement of about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100% of total nucleic acids in single cells. The nucleic acidamplification may result in a global measurement of all nucleic acidsequences in single cells. The nucleic acid amplification may result ina measurement of targeted nucleic acid sequences in single cells bysequencing. The nucleic acid amplification may result in a measurementof targeted nucleic acid sequences in single cells by an array.

The amplification agents may comprise a fixed panel of primers. Theamplification agents may comprise at least one pair of custom primers.The amplification agents may comprise at least one pair of controlprimers. The amplification agents may comprise at least one pair ofhousekeeping gene primers. The amplification agents suitable maycomprise a PCR master mix. The kit may further comprise instructions forprimer design and optimization. The kit may further comprise a microwellplate, wherein the microwell plate may comprise at least one well inwhich no more than one bead is distributed. The kit may further compriseone or more additional containers. The one or more additional containersmay comprise one or more additional plurality of sample tags. Theplurality of one or more additional sample tags in the one or moreadditional containers are different from the first plurality of sampletags in the first container. The one or more additional containers maycomprise one or more additional molecular identifier labels. The one ormore additional molecular identifier labels of the one or moreadditional containers are the same as the one or more additionalmolecular identifier labels of the second container.

The methods and kits disclosed herein may comprise the use of one ormore pipette tips and/or containers (e.g., tubes, vials, multiwellplates, microwell plates, eppendorf tubes, glass slides, beads). In someinstances, the pipet tips are low binding pipet tips. Alternatively, oradditionally, the containers may be low binding containers. Low bindingpipet tips and low binding containers may have reduced leaching and/orsubsequent sample degradation associated with silicone-based tips andnon-low binding containers. Low binding pipet tips and low bindingcontainers may have reduced sample binding as compared to non-lowbinding pipet tips and containers. Examples of low binding tips include,but are not limited to, Corning® DeckWorks™ low binding tips and AvantPremium low binding graduated tips. A non-limiting list of low-bindingcontainers include Corning® Costar® low binding microcentrifuge tubesand Cosmobrand low binding PCR tubes and microcentrifuge tubes.

Any of the kits disclosed herein can further comprise software. Forexample, a kit can comprise software for analyzing sequences, such asbarcodes or target sequences. For example, a kit can comprise softwarefor analyzing sequences, such as barcodes or target sequences forcounting unique target molecules, such as unique target molecules from asingle cell. For example, a kit can comprise software for analyzingsequences, such as barcodes or target sequences for counting uniquetarget molecules, such as unique target molecules from a gene, such as agene from a single cell.

Microwells and Microwell Arrays

In some instances, the methods of the disclosure provide for contactinga solid support comprising a conjugated oligonucleotide with a cell. Thecontacting step may be performed on a surface. Exemplary surfaces mayinclude a microwell, a tube, a flask, and chip. In some instances, thesurface comprises a microwell. In some instances, the microwell is partof a microwell array.

The microwells of a microwell array may be of a size and shape capableof containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cellsper microwell. The microwells may be of a size and shape capable ofcontaining at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells permicrowell. The microwells of a microwell array may be of a size andshape capable of containing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore solid supports per microwell. The microwells may be of a size andshape capable of containing at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore solid supports per microwell. A microwell may comprise at most onecell and one solid support. A microwell may comprise at most one celland two solid supports. A microwell may comprise at least one cell andat most one solid support. A microwell may comprise at least one celland at most two solid supports.

Microwells on the microwell array may be arranged horizontally. Themicrowells may be arranged vertically. The microwells may be arrangedwith equal or near equal spacing. The microwell array may have markersassociated with one or more microwells. For example, the microwells ofthe microwell array may be divided into groups each comprised of aprescribed number of microwells. These groups may be provided on theprincipal surface of the substrate. Markers may be provided so that theposition of each group may be determined. A marker may be detectable bythe naked eye. A marker may be a marker that requires optics to see(e.g., fluorescent marker, emission marker, UV marker).

A microwell array may comprise at least about 96, 384, 1000, 5000,10000, 15000, 100000, 150000, 500000, 1000000, or 5000000 or moremicrowells. A microwell array may comprise at most about 96, 384, 1000,5000, 10000, 15000, 100000, 150000 500000, 1000000, or 5000000 or moremicrowells.

The shape of the microwell may be cylindrical. The shape of themicrowell may be noncylindrical, such as a polyhedron comprised ofmultiple faces (for example, a parallelepiped, hexagonal column, oroctagonal column), an inverted cone, an inverted pyramid (invertedtriangular pyramid, inverted square pyramid, inverted pentagonalpyramid, inverted hexagonal pyramid, or an inverted polygonal pyramidwith seven or more angles). The microwell may comprise a shape combiningtwo or more of these shapes. For example, it may be partly cylindrical,with the remainder having the shape of an inverted cone. The shape ofthe microwell may be one in which a portion of the top of an invertedcone or inverted pyramid is cut off. The mouth of the microwell may beon the top of the microwell or the bottom of the microwell. The bottomof the microwell may be flat, but curved surfaces (e.g., convex orconcave) are also possible. The shape and size of the microwell may bedetermined in consideration of the type of cell and/or solid substrate(e.g., shape, size) to be stored in the microwell.

The diameter of the microwell may refer to the largest circle that maybe inscribed in the planar shape of the microwell. The diameter of themicrowell may be at least about 0.1, 0.5, 1, 2, or 3-fold or more thediameter of the cell and/or solid support to be contained in themicrowell. The diameter of the microwell may be at most about 0.1, 0.5,1, 2, or 3-fold or more the diameter of the cell and/or solid support tobe contained in the microwell. The diameter of the microwell may be atleast about 10, 20, 30, 40, or 50% or more the diameter of the solidsupport. The diameter of the microwell may be at most about 10, 20, 30,40, or 50% or more the diameter of the solid support. The diameter ofthe microwell may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,or 50 or more micrometers. The diameter of the microwell may be at mostabout 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more micrometers. Thediameter of the microwell is about 25 micrometers. In some instances,the diameter of the microwell is about 30 micrometers. In someinstances, the diameter of the microwell is about 28 micrometers.

The difference between the microwell volume and the solid support volumemay be at least about 1×10(⁻¹⁴) m³, 1.5×10(⁻¹⁴) m³, 1.7×10(⁻¹⁴) m³,2.0×10(⁻¹⁴) m³, 2.5×10(⁻¹⁴) m³, or 3.0×10(⁻¹⁴) m³ or more. Thedifference between the microwell volume and the solid support volume maybe at most about 1×10(⁻¹⁴) m³, 1.5×10(⁻¹⁴) m³, 1.7×10(⁻¹⁴) m³,2.0×10(⁻¹⁴) m³, 2.5×10(⁻¹⁴) m³, or 3.0×10(⁻¹⁴) m³ or more. Thedifference between the microwell volume and the solid support volume maybe at least about 1×10(⁻¹¹) L, 1.5×10(⁻¹¹) L, 1.7×10(⁻¹¹) L, 2.0×10(⁻¹¹)L, 2.5×10(⁻¹¹) L, or 3.0×10(⁻¹¹) L or more. The difference between themicrowell volume and the solid support volume may be at most about1×10(⁻¹¹) L, 1.5×10(⁻¹¹) L, 1.7×10(⁻¹¹) L, 2.0×10(⁻¹¹) L, 2.5×10(⁻¹¹) L,or 3.0×10(⁻¹¹) L or more. FIG. 7 illustrates exemplary statistics aboutthe volume of the microwell, the solid support, and the differencesbetween the microwell and the solid support volumes.

The depth of the microwell may be at least about 0.1, 0.5, 1, 2, 3, 4,or 5-fold or more the diameter of the cell and/or solid support to becontained in the microwell. The depth of the microwell may be at mostabout 0.1, 0.5, 1, 2, 3, 4, or 5-fold or more the diameter of the celland/or solid support to be contained in the microwell. The depth of themicrowell may be at least about 10, 20, 30, 40, or 50% or more the depthof the solid support. The depth of the microwell may be at most about10, 20, 30, 40, or 50% or more the depth of the solid support. The depthof the microwell may be at least about 5, 10, 15, 20, 25, 30, 35, 40,45, or 50 or more micrometers. The depth of the microwell may be at mostabout 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more micrometers. Thedepth of the microwell may be about 30 micrometers. The depth of themicrowell may be about 28 micrometers. The microwell may be flat, orsubstantially flat.

A microwell array may comprise spacing between the wells. The spacingbetween the wells may be at least about 5, 10, 25, 20, 25, 30, 35, 40,45, or 50 or more micrometers. The spacing between the wells may be atmost about 5, 10, 25, 20, 25, 30, 35, 40, 45, or 50 or more micrometers.The spacing between the wells may be about 15 micrometers. The spacingbetween the wells may be about 25 micrometers.

There may be differences in the height of dips and rises at any positionon the inner wall of a microwell. By creating dips and rises on aportion of the inner wall of a well that has been treated forsmoothness, functionality may be added to the well. The inner wall of amicrowell may be smoothed by etching. The degree of vacuum in theetching device, the type of etching gas, the etching steps, and the likemay be suitably selected. For example, smoothing of the inner wall of amicrowell may be conducted by wet etching or by combining a hotoxidation step with oxide film etching. The inner wall of the microwellmay be functionalized (e.g., functionalized with an oligonucleotide, areactive group, a functional group).

The microwell array may be made of silicon, metal (e.g., aluminum,stainless steel, copper, nickel, chromium, and titanium), PDMS(elastomer), glass, polypropylene, agarose, gelatin, pluoronic (e.g.,pluronic F127), plastics (e.g., plastics that are naturally hydrophilic,such as PMMA), plastics (e.g., PP, COP, COC) and elastomer (e.g., PDMS)that are hydrophobic but may be treated to be made hydrophilic),hydrogels (e.g., polyacrylamide, alginate), or resin (e.g., polyimide,polyethylene, vinyl chloride, polypropylene, polycarbonate, acrylic, andpolyethylene terephthalate). The microwell array may be made of amaterial that is hydrophobic. The microwell array may be made of amaterial that is hydrophobic but coated to be made hydrophilic (e.g., byoxygen plasma treatment). The microwell array may be made of a materialthat is hydrophilic but coated to be made hydrophobic.

A microwell array may be assembled. Microwell array assembly maycomprise obtaining a silicon wafter with patterning (e.g., patternedposts made with SU8 photoresist) and incubating it with PDMS material tocreate arrays of wells through soft lithography (e.g., at 80 C for a fewhours). For example, uncured PDMS may be liquid. Uncured PDMS may fillgaps between posts. When PDMS is cured by heat, it may be come solid,thereby generating the array of wells. An optical adhesive (e.g.,NOA81/NOA63) may be applied to the PDMS material (e.g., using UV light)to create an array of posts (e.g., a plurality of arrays). Theapplication may be performed for at least about 1 second, 2 seconds, 3seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9seconds, 10 seconds or 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5or more minutes. A layer comprising agarose may be applied to theoptical adhesive. The agarose layer may be at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10% or more agarose. The agarose layer may be most about 1,2, 3, 4 5, 6, 7, 8, 9, 10% or more agarose. The agarose layer may beabout 5% agarose. The agarose layer may be set on Gelbond film, or anyhydrophilic substrate that the agarose may adhere to. The incubation ofthe agarose layer on the optical surface may be at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more minutes. The incubation of the agaroselayer on the optical surface may be at most about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more minutes.

In some instances, the methods of the disclosure may use a surface thatmay not comprise microwells. The surface may be glass, plastic, metal.The surface may be coated with solid supports, extracellular matrix,polymers. The surface may not comprise wells. The surface may comprisesolid supports spatially arranged to limit molecular diffusion. Themethods of the disclosure of capturing cells and/or cell contents mayoccur on a flat surface. The methods of the disclosure of capturingcells and/or cell contents may occur in a suspension.

Cells and Samples

The cell and of the disclosure may be a cell from an animal (e.g.,human, rat, pig, horse, cow, dog, mouse). In some instances, the cell isa human cell. The cell may be a fetal human cell. The fetal human cellmay be obtained from a mother pregnant with the fetus. The cell may be acell from a pregnant mother. The cell may be a cell from a vertebrate,invertebrate, fungi, archae, or bacteria. The cell may be from amulticellular tissue (e.g., an organ (e.g., brain, liver, lung, kidney,prostate, ovary, spleen, lymph node, thyroid, pancreas, heart, skeletalmuscle, intestine, larynx, esophagus, and stomach), a blastocyst). Thecell may be a cell from a cell culture. The cell may be a HeLa cell, aK562 cell, a Ramos cell, a hybridoma, a stem cell, an undifferentiatedcell, a differentiated cell, a circulating cell, a CHO cell, a 3T3 cell,and the like.

In some instances, the cell is a cancerous cell. Non-limiting examplesof cancer cells may include a prostate cancer cell, a breast cancercell, a colon cancer cell, a lung cancer cell, a brain cancer cell, andan ovarian cancer cell. In some instances, the cell is from a cancer(e.g., a circulating tumor cell). Non-limiting examples of cancers mayinclude, adenoma, adenocarcinoma, squamous cell carcinoma, basal cellcarcinoma, small cell carcinoma, large cell undifferentiated carcinoma,chondrosarcoma, and fibrosarcoma.

In some instances, the cell is a rare cell. A rare cell can be acirculatiing tumor cell (CTC), circulating epithelial cell (CEC),circulating stem cell (CSC), stem cells, undifferentiated stem cells,cancer stem cells, bone marrow cells, progenitor cells, foam cells,fetal cells, mesenchymal cells, circulating endothelial cells,circulating endometrial cells, trophoblasts, immune system cells (hostor graft), connective tissue cells, bacteria, fungi, or pathogens (forexample, bacterial or protozoa), microparticles, cellular fragments,proteins and nucleic acids, cellular organelles, other cellularcomponents (for example, mitochondria and nuclei), and viruses.

In some instances, the cell is from a tumor. In some instances, thetumor is benign or malignant. The tumor cell may comprise a metastaticcell. In some instances, the cell is from a solid tissue that comprisesa plurality of different cell types (e.g., different genotypes).

The cell may comprise a virus, bacterium, fungus, and parasite. Virusesmay include, but are not limited to, DNA or RNA animal viruses (e.g.,Picornaviridae (e.g., polioviruses), Reoviridae (e.g., rotaviruses),Togaviridae (e.g., encephalitis viruses, yellow fever virus, rubellavirus), Orthomyxoviridae (e.g., influenza viruses), Paramyxoviridae(e.g., respiratory syncytial virus, measles virus, mumps virus,parainfluenza virus), Rhabdoviridae (e.g., rabies virus), Coronaviridae,Bunyaviridae, Flaviviridae, Filoviridae, Arenaviridae, Bunyaviridae andRetroviridae (e.g., human T cell lymphotropic viruses (HTLV), humanimmunodeficiency viruses (HW), Papovaviridae (e.g., papilloma viruses),Adenoviridae (e.g., adenovirus), Herpesviridae (e.g., herpes simplexviruses), and Poxviridae (e.g., variola viruses)).

Exemplary bacteria that may be used in the methods of the disclosure mayinclude Actinomedurae, Actinomyces israelii, Bacillus anthracis,Bacillus cereus, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacterium,Enterococcus faecalis, Listeria monocytogenes, Nocardia,Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epiderm,Streptococcus mutans, Streptococcus pneumoniae and the like. Gramnegative bacteria include, but are not limited to, Afipia felis,Bacteroides, Bartonella bacilliformis, Bortadella pertussis, Borreliaburgdorferi, Borrelia recurrentis, Brucella, Calymmatobacteriumgranulomatis, Campylobacter, Escherichia coli, Francisella tularensis,Gardnerella vaginalis, Haemophilius aegyptius, Haemophilius ducreyi,Haemophilius influenziae, Heliobacter pylori, Legionella pneumophila,Leptospira interrogans, Neisseria meningitidia, Porphyromonasgingivalis, Providencia sturti, Pseudomonas aeruginosa, Salmonellaenteridis, Salmonella typhi, Serratia marcescens, Shigella boydii,Streptobacillus moniliformis, Streptococcus pyogenes, Treponemapallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis andthe like. Other bacteria may include Myobacterium avium, Myobacteriumleprae, Myobacterium tuberculosis, Bartonella henseiae, Chlamydiapsittaci, Chlamydia trachomatis, Coxiella burnetii, Mycoplasmapneumoniae, Rickettsia akari, Rickettsia prowazekii, Rickettsiarickettsii, Rickettsia tsutsugamushi, Rickettsia typhi, Ureaplasmaurealyticum, Diplococcus pneumoniae, Ehrlichia chafensis, Enterococcusfaecium, Meningococci and the like.

Exemplary fungi to be used in the methods of the disclosure may include,but are not limited to Aspergilli, Candidae, Candida albicans,Coccidioides immitis, Cryptococci, and combinations thereof.

Exemplary parasites to be used in the methods of the disclosure mayinclude, but are not limited to, Balantidium coli, Cryptosporidiumparvum, Cyclospora cayatanensis, Encephalitozoa, Entamoeba histolytica,Enterocytozoon bieneusi, Giardia lamblia, Leishmaniae, Plasmodii,Toxoplasma gondii, Trypanosomae, trapezoidal amoeba, worms (e.g.,helminthes), particularly parasitic worms including, but not limited to,Nematoda (roundworms, e.g., whipworms, hookworms, pinworms, ascarids,filarids and the like), Cestoda (e.g., tapeworms).

The sample of the disclosure may be a sample from an animal (e.g.,human, rat, pig, horse, cow, dog, mouse). In some instances, the sampleis a human sample. The sample may be a fetal human sample. The fetalhuman sample may be obtained from a mother pregnant with the fetus. Thesample may be a sample from a pregnant mother. The sample may be asample from a vertebrate, invertebrate, fungi, archae, or bacteria. Thesample may be from a multicellular tissue (e.g., an organ (e.g., brain,liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid,pancreas, heart, skeletal muscle, intestine, larynx, esophagus, andstomach), a blastocyst). The sample may be a cell from a cell culture.

The sample may comprise a plurality of cells. The sample may comprise aplurality of the same type of cell. The sample may comprise a pluralityof different types of cells. The sample may comprise a plurality ofcells at the same point in the cell cycle and/or differentiationpathway. The sample may comprise a plurality of cells at differentpoints in the cell cycle and/or differentiation pathway. A sample maycomprise a plurality of samples.

The plurality of samples may comprise at least 5, 10, 20, 30, 40, 50,60, 70, 80, 90 or 100 or more samples. The plurality of samples maycomprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900 or1000 or more samples. The plurality of samples may comprise at leastabout 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 samples, 9000, or10,000 samples, or 100,000 samples, or 1,000,000 or more samples. Theplurality of samples may comprise at least about 10,000 samples.

The one or more nucleic acids in the first sample may be different fromone or more nucleic acids in the second sample. The one or more nucleicacids in the first sample may be different from one or more nucleicacids in a plurality of samples. The one or more nucleic acids maycomprise a length of at least about 1 nucleotide, 2 nucleotides, 5nucleotides, 10 nucleotides, 20 nucleotides, 50 nucleotides, 100nucleotides, 200 nucleotides, 300 nucleotides, 500 nucleotides, 1000nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, 5000nucleotides, 10,000 nucleotides, 100,000 nucleotides, 1,000,000nucleotides.

The first sample may comprise one or more cells and the second samplemay comprise one or more cells. The one or more cells of the firstsample may be of the same cell type as the one or more cells of thesecond sample. The one or more cells of the first sample may be of adifferent cell type as one or more different cells of the plurality ofsamples. The cell type may be chondrocyte, osteoclast, adipocyte,myoblast, stem cell, endothelial cell or smooth muscle cell. The celltype may be an immune cell type. The immune cell type may be a T cell, Bcell, thrombocyte, dendritic cell, neutrophil, macrophage or monocyte.

The plurality of samples may comprise one or more malignant cell. Theone or more malignant cells may be derived from a tumor, sarcoma orleukemia.

The plurality of samples may comprise at least one bodily fluid. Thebodily fluid may comprise blood, urine, lymphatic fluid, saliva. Theplurality of samples may comprise at least one blood sample.

The plurality of samples may comprise at least one cell from one or morebiological tissues. The one or more biological tissues may be a bone,heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine,liver, pancreas, spleen, kidney, gall bladder, thyroid gland, adrenalgland, mammary gland, ovary, prostate gland, testicle, skin, adipose,eye or brain.

The biological tissue may comprise an infected tissue, diseased tissue,malignant tissue, calcified tissue or healthy tissue.

The plurality of samples may be from one or more sources. The pluralityof samples may be from two or more sources. The plurality of samples maybe from one or more subjects. The plurality of samples may be from twoor more subjects. The plurality of samples may be from the same subject.The one or more subjects may be from the same species. The one or moresubjects may be from different species. The one or more subjects may behealthy. The one or more subjects may be affected by a disease, disorderor condition. The plurality of samples may comprise cells of an originselected from a mammal, bacteria, virus, fungus or plant. The one ormore samples may be from a human, horse, cow, chicken, pig, rat, mouse,monkey, rabbit, guinea pig, sheep, goat, dog, cat, bird, fish, frog andfruit fly.

The plurality of samples may be obtained concurrently. The plurality ofsamples may be obtained at the same time. The plurality of samples maybe obtained sequentially. The plurality of samples may be obtained overa course of years, 100 years, 10 years, 5 years, 4 years, 3 years, 2years or 1 year of obtaining one or more different samples. One or moresamples may be obtained within about one year of obtaining one or moredifferent samples. One or more samples may be obtained within 12 months,11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 4 months,3 months, 2 months or 1 month of obtaining one or more differentsamples. One or more samples may be obtained within 30 days, 28 days, 26days, 24 days, 21 days, 20 days, 18 days, 17 days, 16 days, 15 days, 14days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6days, 5 days, 4 days, 3 days, 2 days or one day of obtaining one or moredifferent samples. One or more samples may be obtained within about 24hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour of obtaining one ormore different samples. One or more samples may be obtained within about60 sec, 45 sec, 30 sec, 20 sec, 10 sec, 5 sec, 2 sec or 1 sec ofobtaining one or more different samples. One or more samples may beobtained within less than one second of obtaining one or more differentsamples.

Target Molecules

The methods and kits disclosed herein may be used in the stochasticlabeling of molecules. Such molecules include, but are not limited to,polynucleotides and polypeptides. As used herein, the terms“polynucleotide” and “nucleic acid molecule” refers to a polymeric formof nucleotides of any length, either ribonucleotides,deoxyribonucleotides, locked nucleic acids (LNA) or peptide nucleicacids (PNAs), that comprise purine and pyrimidine bases, or othernatural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. A “polynucleotide” or “nucleic acidmolecule” may consist of a single nucleotide or base pair.Alternatively, the “polynucleotide” or “nucleic acid molecule” comprisestwo or more nucleotides or base pairs. For example, the “polynucleotide”or “nucleic acid molecule” comprises at least about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides orbase pairs. In another example, the polynucleotide comprises at leastabout 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 8500, 9000, 9500, or 10000 nucleotides or base pairs.The backbone of the polynucleotide may comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleoside sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes may be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired. In some instances, themolecules are DNA, RNA, or DNA-RNA hybrids. The molecules may besingle-stranded or double-stranded. In some instances, the molecules areRNA molecules, such as mRNA, rRNA, tRNA, ncRNA, lncRNA, siRNA, microRNAor miRNA. The RNA molecules may be polyadenylated. Alternatively, themRNA molecules are not polyadenylated. Alternatively, the molecules areDNA molecules. The DNA molecules may be genomic DNA. The DNA moleculesmay comprise exons, introns, untranslated regions, or any combinationthereof. In some instances, the molecules are a panel of molecules.

The methods and kits disclosed herein may be used to stochasticallylabel individual occurrences of identical or nearly identical moleculesand/or different molecules. In some instances, the methods and kitsdisclosed herein may be used to stochastically label identical or nearlyidentical molecules (e.g., molecules comprise identical or nearlyidentical sequences). For example, the molecules to be labeled compriseat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity. The nearly identical molecules may differ by less than about100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 nucleotide or base pair. The plurality of nucleic acids in one ormore samples of the plurality of samples may comprise two or moreidentical sequences. At least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, or 100% of the total nucleic acids in one ormore of the plurality of samples may comprise the same sequence. Theplurality of nucleic acids in one or more samples of the plurality ofsamples may comprise at least two different sequences. At least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the total nucleic acids inone or more of the plurality of samples may comprise at least twodifferent sequences. In some instances, the molecules to be labeled arevariants of each other. For example, the molecules to be labeled maycontain single nucleotide polymorphisms or other types of mutations. Inanother example, the molecules to be labeled are splice variants. Insome instances, at least one molecule is stochastically labeled. Inother instances, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 identical ornearly identical molecules are stochastically labeled. Alternatively, atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000 identical or nearly identical molecules arestochastically labeled. In other instances, at least 1500; 2,000; 2500;3,000; 3500; 4,000; 4500; 5,000; 6,000; 7,000; 8,000; 9,000; or 10000identical or nearly identical molecules are stochastically labeled. Inother instances; at least 15,000; 20,000; 25,000; 30,000; 35,000;40,000; 45,000; 50,000; 60,000; 70,000; 80,000; 90,000; or 100,000identical or nearly identical molecules are stochastically labeled.

In other instances, the methods and kits disclosed herein may be used tostochastically label different molecules. For example, the molecules tobe labeled comprise less than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity. Thedifferent molecules may differ by at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotidesor base pairs. In some instances, at least one molecule isstochastically labeled. In other instances, at least 2, 3, 4, 5, 6, 7,8, 9, or 10 different molecules are stochastically labeled.Alternatively, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 different molecules arestochastically labeled. In other instances, at least 1500; 2,000; 2500;3,000; 3500; 4,000; 4500; 5,000; 6,000; 7,000; 8,000; 9,000; or 10000different molecules are stochastically labeled. In other instances; atleast 15,000; 20,000; 25,000; 30,000; 35,000; 40,000; 45,000; 50,000;60,000; 70,000; 80,000; 90,000; or 100,000 different molecules arestochastically labeled.

The different molecules to be labeled may be present in the sample atdifferent concentrations or amounts. For example, the concentration oramount of one molecule is greater than the concentration or amount ofanother molecule in the sample. In some instances, the concentration oramount of at least one molecule in the sample is at least about 1.5, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100 or more times greater than the concentration oramount of at least one other molecule in the sample. In some instances,the concentration or amount of at least one molecule in the sample is atleast about 1000 or more times greater than the concentration or amountof at least one other molecule in the sample. In another example, theconcentration or amount of one molecule is less than the concentrationor amount of another molecule in the sample. The concentration or amountof at least one molecule in the sample may be at least about 1.5, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100 or more times less than the concentration oramount of at least one other molecule in the sample. The concentrationor amount of at least one molecule in the sample may be at least about1000 or more times less than the concentration or amount of at least oneother molecule in the sample.

In some instances, the molecules to be labeled are in one or moresamples. The molecules to be labeled may be in two or more samples. Thetwo or more samples may contain different amounts or concentrations ofthe molecules to be labeled. In some instances, the concentration oramount of one molecule in one sample may be greater than theconcentration or amount of the same molecule in a different sample. Forexample, a blood sample might contain a higher amount of a particularmolecule than a urine sample. Alternatively, a single sample is dividedinto two or more subsamples. The subsamples may contain differentamounts or concentrations of the same molecule. The concentration oramount of at least one molecule in one sample may be at least about 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 or more times greater than the concentrationor amount of the same molecule in another sample. Alternatively, theconcentration or amount of one molecule in one sample may be less thanthe concentration or amount of the same molecule in a different sample.For example, a heart tissue sample might contain a higher amount of aparticular molecule than a lung tissue sample. The concentration oramount of at least one molecule in one sample may be at least about 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 or more times less than the concentration oramount of the same molecule in another sample. In some instances, thedifferent concentrations or amounts of a molecule in two or moredifferent samples is referred to as sample bias.

The methods and kits disclosed herein may be used for the analysis oftwo or more molecules from two or more samples. The two or moremolecules may comprise two or more polypeptides. The method may comprisedetermining the identity of two or more labeled polypeptides.Determining the identity of two or more labeled polypeptides maycomprise mass spectrometry. The method may further comprise combiningthe labeled polypeptides of the first sample with the labeledpolypeptides of the second sample. The labeled polypeptides may becombined prior to determining the number of different labeledpolypeptides. The method may further comprise combining the firstsample-tagged polypeptides and the second sample-tagged polypeptides.The first sample-tagged polypeptides and the second sample-taggedpolypeptides may be combined prior to contact with the plurality ofmolecular identifier labels. Determining the number of different labeledpolypeptides may comprise detecting at least a portion of the labeledpolypeptide. Detecting at least a portion of the labeled polypeptide maycomprise detecting at least a portion of the sample tag, molecularidentifier label, polypeptide, or a combination thereof.

As used herein, the term “polypeptide” refers to a molecule comprisingat least one peptide. In some instances, the polypeptide consists of asingle peptide. Alternatively, the polypeptide comprises two or morepeptides. For example, the polypeptide comprises at least about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000peptides. Examples of polypeptides include, but are not limited to,amino acids, proteins, peptides, hormones, oligosaccharides, lipids,glycolipids, phospholipids, antibodies, enzymes, kinases, receptors,transcription factors, and ligands.

Subjects

The methods and kits disclosed herein may comprise use of a cell orsample from one or more subjects. A subject may be a human or anon-human subject. A subject may be living. A subject may be dead. Asubject may be a human that is under the care of a caregiver (e.g.,medical professional). A subject may be suspected of having a disease. Asubject may have a disease. A subject may have symptoms of a disease. Asubject may be a subject that provides one or more samples. A subjectmay be a mammal, reptile, amphibian, and/or bird. A subject may be anon-human primate.

Enzymes

The methods and kits disclosed herein may comprise one or more enzymes.Examples of enzymes include, but are not limited to ligases, reversetranscriptases, polymerases, and restriction nucleases. In someinstances, attachment of the oligonucleotide tag to the moleculescomprises the use of one or more ligases. Examples of ligases include,but are not limited to, DNA ligases such as DNA ligase I, DNA ligaseIII, DNA ligase W, and T4 DNA ligase, and RNA ligases such as T4 RNAligase I and T4 RNA ligase II.

The methods and kits disclosed herein may further comprise the use ofone or more reverse transcriptases. In some instances, the reversetranscriptase is a HIV-1 reverse transcriptase, M-MLV reversetranscriptase, AMV reverse transcriptase, and telomerase reversetranscriptase. In some instances, the reverse transcriptase is M-MLVreverse transcriptase.

In some instances, the methods and kits disclosed herein comprise theuse of one or more polymerases. Examples of polymerases include, but arenot limited to, DNA polymerases and RNA polymerases. In some instances,the DNA polymerase is a DNA polymerase I, DNA polymerase II, DNApolymerase III holoenzyme, and DNA polymerase W. Commercially availableDNA polymerases include, but are not limited to, Bst 2.0 DNA Polymerase,Bst 2.0 WarmStart™ DNA Polymerase, Bst DNA Polymerase, Sulfolobus DNAPolymerase IV, Taq DNA Polymerase, 9° N™m DNA Polymerase, Deep VentR™(exo-) DNA Polymerase, Deep VentR™ DNA Polymerase, Hemo KlenTaq™,LongAmp® Taq DNA Polymerase, OneTaq® DNA Polymerase, Phusion® DNAPolymerase, Q5™ High-Fidelity DNA Polymerase, Therminator™. y DNAPolymerase, Therminator™ DNA Polymerase, Therminator™ II DNA Polymerase,Therminator™ III DNA Polymerase, VentR® DNA Polymerase, VentR® (exo-)DNA Polymerase, Bsu DNA Polymerase, phi29 DNA Polymerase, T4 DNAPolymerase, T7 DNA Polymerase, Terminal Transferase, Titanium® TaqPolymerase, KAPA Taq DNA Polymerase and KAPA Taq Hot Start DNAPolymerase.

Alternatively, the polymerase is an RNA polymerases such as RNApolymerase I, RNA polymerase II, RNA polymerase III, E. coli Poly(A)polymerase, phi6 RNA polymerase (RdRP), Poly(U) polymerase, SP6 RNApolymerase, and T7 RNA polymerase.

In some instances, the methods and kits disclosed herein comprise one ormore restriction enzymes. Restriction enzymes include type I, type II,type III, and type W restriction enzymes. In some instances, Type Ienzymes are complex, multi-subunit, combinationrestriction-and-modification enzymes that cut DNA at random far fromtheir recognition sequences. Generally, type II enzymes cut DNA atdefined positions close to or within their recognition sequences. Theymay produce discrete restriction fragments and distinct gel bandingpatterns. Type III enzymes are also large combinationrestriction-and-modification enzymes. They often cleave outside of theirrecognition sequences and may require two such sequences in oppositeorientations within the same DNA molecule to accomplish cleavage; theyrarely give complete digests. In some instances, type W enzymesrecognize modified, typically methylated DNA and may be exemplified bythe McrBC and Mrr systems of E. coli.

Additional Reagents

The methods and kits disclosed herein may comprise the use of one ormore reagents. Examples of reagents include, but are not limited to, PCRreagents, ligation reagents, reverse transcription reagents, enzymereagents, hybridization reagents, sample preparation reagents, andreagents for nucleic acid purification and/or isolation.

The methods and kits disclosed herein may comprise the use of one ormore buffers. Examples of buffers include, but are not limited to, washbuffers, ligation buffers, hybridization buffers, amplification buffers,and reverse transcription buffers. In some instances, the hybridizationbuffer is a commercially available buffer, such as TMAC Hyb solution,SSPE hybridization solution, and ECONO™ hybridization buffer. Thebuffers disclosed herein may comprise one or more detergents.

The methods and kits disclosed herein may comprise the use of one ormore carriers. Carriers may enhance or improve the efficiency of one ormore reactions disclosed herein (e.g., ligation reaction, reversetranscription, amplification, hybridization). Carriers may decrease orprevent non-specific loss of the molecules or any products thereof(e.g., labeled-molecule, labeled-cDNA molecule, labeled-amplicon). Forexample, the carrier may decrease non-specific loss of alabeled-molecule through absorption to surfaces. The carrier maydecrease the affinity of the molecule, labeled-molecule, or any productthereof to a surface or substrate (e.g., container, eppendorf tube,pipet tip). Alternatively, the carrier may increase the affinity of themolecule or any product thereof to a surface or substrate (e.g., bead,array, glass, slide, chip). Carriers may protect the molecule or anyproduct thereof from degradation. For example, carriers may protect anRNA molecule or any product thereof from ribonucleases. Alternatively,carriers may protect a DNA molecule or any product thereof from a DNase.Examples of carriers include, but are not limited to, nucleic acidmolecules such as DNA and/or RNA, or polypeptides. Examples of DNAcarriers include plasmids, vectors, polyadenylated DNA, and DNAoligonucleotides. Examples of RNA carriers include polyadenylated RNA,phage RNA, phage MS2 RNA, E. coli RNA, yeast RNA, yeast tRNA, mammalianRNA, mammalian tRNA, short polyadenylated synthetic ribonucleotides andRNA oligonucleotides. The RNA carrier may be a polyadenylated RNA.Alternatively, the RNA carrier may be a non-polyadenylated RNA. In someinstances, the carrier is from a bacteria, yeast, or virus. For example,the carrier may be a nucleic acid molecule or a polypeptide derived froma bacteria, yeast or virus. For example, the carrier is a protein fromBacillus subtilis. In another example, the carrier is a nucleic acidmolecule from Escherichia coli. Alternatively, the carrier is a nucleicacid molecule or peptide from a mammal (e.g., human, mouse, goat, rat,cow, sheep, pig, dog, or rabbit), avian, amphibian, or reptile.

The methods and kits disclosed herein may comprise the use of one ormore control agents. Control agents may include control oligos, inactiveenzymes, non-specific competitors. Alternatively, the control agentscomprise bright hybridization, bright probe controls, nucleic acidtemplates, spike-in controls, PCR amplification controls. The PCRamplification controls may be positive controls. In other instances, thePCR amplification controls are negative controls. The nucleic acidtemplate controls may be of known concentrations. The control agents maycomprise one or more labels.

Spike-in controls may be templates that are added to a reaction orsample. For example, a spike-in template may be added to anamplification reaction. The spike-in template may be added to theamplification reaction any time after the first amplification cycle. Insome instances, the spike-in template is added to the amplificationreaction after the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th,12th, 13th, 14th, 15th, 20th, 25th, 30th, 35th, 40th, 45th, or 50thamplification cycle. The spike-in template may be added to theamplification reaction any time before the last amplification cycle. Thespike-in template may comprise one or more nucleotides or nucleic acidbase pairs. The spike-in template may comprise DNA, RNA, or anycombination thereof. The spike-in template may comprise one or morelabels.

Detectable Labels

The methods, kits, and compositions disclosed herein may furthercomprise a detectable label. The terms “detectable label”, “tag” or“label” may be used interchangeably and refer to any chemical moietyattached to a molecule (e.g., nucleotide, nucleotide polymer, or nucleicacid binding factor, molecular barcode). The chemical moiety may becovalently attached the molecule. The chemical moiety may benon-covalently attached to the molecule. The molecular barcodes, sampletags and molecular identifier labels may further comprise a detectablelabel, tag or label. Preferably, the label is detectable and renders thenucleotide or nucleotide polymer detectable to the practitioner of theinvention. Detectable labels that may be used in combination with themethods disclosed herein include, for example, a fluorescent label, achemiluminescent label, a quencher, a radioactive label, biotin, pyrenemoiety, gold, or combinations thereof. Non-limiting example ofdetectable labels include luminescent molecules, fluorochromes,fluorescent quenching agents, colored molecules, radioisotopes orscintillants.

In some instances, the methods disclosed herein further compriseattaching one or more detectable labels to the molecular barcode,molecular identifier label, the sample tag, the labeled nucleic acid orany product thereof (e.g., labeled-amplicon). The methods may compriseattaching two or more detectable labels to the molecular barcode,molecular identifier label, the sample tag or the labeled nucleic acid.Alternatively, the method comprises attaching at least about 3, 4, 5, 6,7, 8, 9, or 10 detectable labels to the molecular barcode, molecularidentifier label, the sample tag or the labeled nucleic acid. In someinstances, the detectable label is a Cy™ label. The Cy™ label is a Cy3label. Alternatively, or additionally, the detectable label is biotin.In some embodiments the detectable label is attached to a probe whichbinds to the molecular barcode, molecular identifier label, the sampletag or the labeled nucleic acid. This may occur, for example, after thenucleic acid or labeled nucleic acid has been hybridized to an array. Inone example the nucleic acid or labeled nucleic acid is bound topartners on an array. After the binding, a probe which may bind thelabeled nucleic acid is bound to the molecules on the array. Thisprocess may be repeated with multiple probes and labels to decrease thelikelihood that a signal is the result of nonspecific binding of a labelor nonspecific binding of the molecule to the array.

A donor acceptor pair may be used as the detectable labels. Either thedonor or acceptor may be attached to a probe that binds a nucleic acid.The probe may be, for example, a nucleic acid probe that may bind to anucleic acid or the labeled nucleic acid. The corresponding donor oracceptor may be added to cause a signal.

In some instances, the detectable label is a Freedom dye, Alexa Fluor®dye, Cy™ dye, fluorescein dye, or LI-COR IRDyes®. In some instances, theFreedom dye is fluorescein (6-FAM™, 6-carboxyfluoroscein), MAX (NHSEster), TYE™ 563, TEX 615, TYE™ 665, TYE 705. The detectable label maybe an Alexa Fluor dye. Examples of Alexa Fluor® dyes include AlexaFluor® 488 (NHS Ester), Alexa Fluor® 532 (NHS Ester), Alexa Fluor® 546(NHS Ester), Alexa Fluor® 594 (NHS Ester), Alexa Fluor® 647 (NHS Ester),Alexa Fluor® 660 (NHS Ester), or Alexa Fluor® 750 (NHS Ester).Alternatively, the detectable label is a Cy™ dye. Examples of Cy™ dyesinclude, but are not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, andCy7. In some instances, the detectable label is a fluorescein dye.Non-limiting examples of fluorescein dyes include 6-FAM™ (Azide), 6-FAM™(NHS Ester), Fluorescein dT, JOE (NHS Ester), TET™, and HEX™. In someinstances, the detectable label is a LI-COR IRDyes®, such as 5′ IRDye®700, 5′ IRDye® 800, or IRDye® 800CW (NHS Ester). In some instances, thedetectable label is TYE™ 563. Alternatively, the detectable label isCy3.

The detectable label may be Rhodamine dye. Examples of rhodamine dyesinclude, but are not limited to, Rhodamine Green™-X (NHS Ester), TAMRA™,TAMRA™ (NHS Ester), Rhodamine Red™-X(NHS Ester), ROX™ (NHS Ester), and5′TAMRA™ (Azide). In other instances, the detectable label is a WellREDDye. WellRED Dyes include, but are not limited to, WellRED D4 dye,WellRED D3 dye, and WellRED D2 dye. In some instances, the detectablelabel is Texas Red®-X (NHS Ester), Lightcycler® 640 (NHS Ester), or Dy750 (NHS Ester).

In some instances, detectable labels include a linker molecule. Examplesof linker molecules include, but are not limited to, biotin, avidin,streptavidin, HRP, protein A, protein G, antibodies or fragmentsthereof, Grb2, polyhistidine, Ni2+, FLAG tags, myc tags. Alternatively,detectable labels include heavy metals, electron donors/acceptors,acridinium esters, dyes and calorimetric substrates. In other instances,detectable labels include enzymes such as alkaline phosphatase,peroxidase and luciferase.

A change in mass may be considered a detectable label, as is the case ofsurface plasmon resonance detection. The skilled artisan would readilyrecognize useful detectable labels that are not mentioned herein, whichmay be employed in the operation of the present invention.

In some instances, detectable labels are used with primers. For example,the universal primer is a labeled with the detectable label (e.g., Cy3labeled universal primer, fluorophore labeled universal primer).Alternatively, the target specific primer is labeled with the detectablelabel (e.g., TYE 563-labeled target specific primer). In otherinstances, detectable labels are used with the sample tags or molecularidentifier labels. For example, the oligonucleotide tag is labeled witha detectable label (e.g., biotin-labeled oligonucleotide tag). In otherinstances, detectable labels are used with the nucleic acid templatemolecule. Detectable labels may be used to detect the labeled-moleculesor labeled-amplicons. Alternatively, detectable labels are used todetect the nucleic acid template molecule.

In some instances, the detectable label is attached to the primer,molecular barcode, sample tag, molecular identifier label,labeled-molecule, labeled-amplicon, probe, HCR probe, and/or non-labelednucleic acid. Methods for attaching the detectable label to the primer,oligonucleotide tag, labeled-molecule, labeled-amplicon, and/ornon-labeled nucleic acid include, but are not limited to, chemicallabeling and enzymatic labeling. In some instances, the detectable labelis attached by chemical labeling. In some embodiments, chemical labelingtechniques comprise a chemically reactive group. Non-limiting examplesof reactive groups include amine-reactive succinimidyl esters such asNHS-fluorescein or NHS-rhodamine, amine-reactive isothiocyanatederivatives including FITC, and sulfhydryl-reactive maleimide-activatedfluors such as fluorescein-5-maleimide. In some embodiments, reaction ofany of these reactive dyes with another molecule results in a stablecovalent bond formed between a fluorophore and the linker and/or agent.In some embodiments, the reactive group is isothiocyanates. In someembodiments, a label is attached to an agent through the primary aminesof lysine side chains. In some embodiments, chemical labeling comprisesa NHS-ester chemistry method.

Alternatively, the detectable label is attached by enzymatic labeling.Enzymatic labeling methods may include, but are not limited to, a biotinacceptor peptide/biotin ligase (AP/Bir A), acyl carrierprotein/phosphopantetheine transferase (ACP/PPTase), human06-alkylguanine transferase (hAGT), Q-tag/transglutaminase (TGase),aldehyde tag/formylglycine-generating enzyme, mutated prokaryoticdehalogenase (HaloTag™), and farnesylation motif/proteinfarnesyltransferase (PFTase) methods. Affinity labeling may include, butis not limited to, noncovalent methods utilizing dihydrofolate reductase(DHFR) and Phe36Val mutant of FK506-binding protein 12 (FKBP12(F36V)),and metal-chelation methods.

Crosslinking reagents may be used to attach a detectable label to theprimer, oligonucleotide tag, labeled-molecule, labeled-amplicon, and/ornon-labeled nucleic acid. In some instances, the crosslinking reagent isglutaraldehyde. Glutaraldehyde may react with amine groups to createcrosslinks by several routes. For example, under reducing conditions,the aldehydes on both ends of glutaraldehyde couple with amines to formsecondary amine linkages.

In some instances, attachment of the detectable label to the primer,oligonucleotide tag, labeled-molecule, labeled-amplicon, and/ornon-labeled nucleic acid comprises periodate-activation followed byreductive amination. In some instances, Sulfo-SMCC or otherheterobifunctional crosslinkers are used to conjugate the detectable tothe primer, oligonucleotide tag, labeled-molecule, labeled-amplicon,and/or non-labeled nucleic acid. For example, Sulfo-SMCC is used toconjugate an enzyme to a drug. In some embodiments, the enzyme isactivated and purified in one step and then conjugated to the drug in asecond step. In some embodiments, the directionality of crosslinking islimited to one specific orientation (e.g., amines on the enzyme tosulfhydryl groups on the antibody).

Diseases/Conditions

Disclosed herein are methods, kits and compositions for diagnosing,monitoring, and/or prognosing a status or outcome of a disease orcondition in a subject. Generally, the method comprises (a)stochastically labeling two or more molecules from two or more samplesto produce two or more labeled nucleic acids; (b) detecting and/orquantifying the two or more labeled nucleic acids; and (c) diagnosing,monitoring, and/or prognosing a status or outcome of a disease orcondition in a subject based on the detecting and/or quantifying of thetwo or more labeled nucleic acids. may The method may further comprisedetermining a therapeutic regimen. The two or more of samples maycomprise one or more samples from a subject suffering from a disease orcondition. The two or more samples may comprise one or more samples froma healthy subject. The two or more samples may comprise one or moresamples from a control.

Monitoring a disease or condition may further comprise monitoring atherapeutic regimen. Monitoring a therapeutic regimen may comprisedetermining the efficacy of a therapeutic regimen. In some instances,monitoring a therapeutic regimen comprises administrating, terminating,adding, or altering a therapeutic regimen. Altering a therapeuticregimen may comprise increasing or reducing the dosage, dosingfrequency, or mode of administration of a therapeutic regimen. Atherapeutic regimen may comprise one or more therapeutic drugs. Thetherapeutic drugs may be an anticancer drug, antiviral drug,antibacterial drug, antipathogenic drug, or any combination thereof.

Cancer

In some instances, the disease or condition is a cancer. The moleculesto be stochastically labeled may be from a cancerous cell or tissue. Insome instances, the cancer is a sarcoma, carcinoma, lymphoma orleukemia. Sarcomas are cancers of the bone, cartilage, fat, muscle,blood vessels, or other connective or supportive tissue. Sarcomasinclude, but are not limited to, bone cancer, fibrosarcoma,chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma,malignant schwannoma, bilateral vestibular schwannoma, osteosarcoma,soft tissue sarcomas (e.g., alveolar soft part sarcoma, angiosarcoma,cystosarcoma phylloides, dermatofibrosarcoma, desmoid tumor, epithelioidsarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma,hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma,neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).

Carcinomas are cancers that begin in the epithelial cells, which arecells that cover the surface of the body, produce hormones, and make upglands. By way of non-limiting example, carcinomas include breastcancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer,rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostatecancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer,vulvar cancer, uterine cancer, oral cancer, penile cancer, testicularcancer, esophageal cancer, skin cancer, cancer of the fallopian tubes,head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma,cutaneous or intraocular melanoma, cancer of the anal region, cancer ofthe small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, cancer of the urethra, cancer of the renal pelvis, cancer of theureter, cancer of the endometrium, cancer of the cervix, cancer of thepituitary gland, neoplasms of the central nervous system (CNS), primaryCNS lymphoma, brain stem glioma, and spinal axis tumors. In someinstances, the cancer is a skin cancer, such as a basal cell carcinoma,squamous cell carcinoma, melanoma, nonmelanoma, or actinic (solar)keratosis.

In some instances, the cancer is a lung cancer. Lung cancer may start inthe airways that branch off the trachea to supply the lungs (bronchi) orthe small air sacs of the lung (the alveoli). Lung cancers includenon-small cell lung carcinoma (NSCLC), small cell lung carcinoma, andmesotheliomia. Examples of NSCLC include squamous cell carcinoma,adenocarcinoma, and large cell carcinoma. The mesothelioma may be acancerous tumor of the lining of the lung and chest cavity (pleura) orlining of the abdomen (peritoneum). The mesothelioma may be due toasbestos exposure. The cancer may be a brain cancer, such as aglioblastoma.

Alternatively, the cancer may be a central nervous system (CNS) tumor.CNS tumors may be classified as gliomas or nongliomas. The glioma may bemalignant glioma, high grade glioma, diffuse intrinsic pontine glioma.Examples of gliomas include astrocytomas, oligodendrogliomas (ormixtures of oligodendroglioma and astocytoma elements), and ependymomas.Astrocytomas include, but are not limited to, low-grade astrocytomas,anaplastic astrocytomas, glioblastoma multiforme, pilocytic astrocytoma,pleomorphic xanthoastrocytoma, and subependymal giant cell astrocytoma.Oligodendrogliomas include low-grade oligodendrogliomas (oroligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomasinclude meningiomas, pituitary adenomas, primary CNS lymphomas, andmedulloblastomas. In some instances, the cancer is a meningioma.

The leukemia may be an acute lymphocytic leukemia, acute myelocyticleukemia, chronic lymphocytic leukemia, or chronic myelocytic leukemia.Additional types of leukemias include hairy cell leukemia, chronicmyelomonocytic leukemia, and juvenile myelomonocytic leukemia.

Lymphomas are cancers of the lymphocytes and may develop from either Bor T lymphocytes. The two major types of lymphoma are Hodgkin'slymphoma, previously known as Hodgkin's disease, and non-Hodgkin'slymphoma. Hodgkin's lymphoma is marked by the presence of theReed-Sternberg cell. Non-Hodgkin's lymphomas are all lymphomas which arenot Hodgkin's lymphoma. Non-Hodgkin lymphomas may be indolent lymphomasand aggressive lymphomas. Non-Hodgkin's lymphomas include, but are notlimited to, diffuse large B cell lymphoma, follicular lymphoma,mucosa-associated lymphatic tissue lymphoma (MALT), small celllymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodalmarginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma(SMZL), extranodal marginal zone B cell lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, and lymphomatoidgranulomatosis.

Pathogenic Infection

In some instances, the disease or condition is a pathogenic infection.The molecules to be stochastically labeled may be from a pathogen. Thepathogen may be a virus, bacterium, fungi, or protozoan. In someinstances, the pathogen may be a protozoan, such as Acanthamoeba (e.g.,A. astronyxis, A. castellanii, A. culbertsoni, A. hatchetti, A.polyphaga, A. rhysodes, A. healyi, A. divionensis), Brachiola (e.g., B.connori, B. vesicularum), Cryptosporidium (e.g., C. parvum), Cyclospora(e.g., C. cayetanensis), Encephalitozoon (e.g., E. cuniculi, E. hellem,E. intestinalis), Entamoeba (e.g., E. histolytica), Enterocytozoon(e.g., E. bieneusi), Giardia (e.g., G. lamblia), Isospora (e.g., I.belli), Microsporidium (e.g., M. africanum, M. ceylonensis), Naegleria(e.g., N. fowleri), Nosema (e.g., N. algerae, N. ocularum),Pleistophora, Trachipleistophora (e.g., T. anthropophthera, T. hominis),and Vittaforma (e.g., V. corneae). The pathogen may be a fungus, suchas, Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, andStachybotrys.

The pathogen may be a bacterium. Exemplary bacteria include, but are notlimited to, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia,Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio,or Yersinia.

The virus may be a reverse transcribing virus. Examples of reversetranscribing viruses include, but are not limited to, single strandedRNA-RT (ssRNA-RT) virus and double-stranded DNA-RT (dsDNA-RT) virus.Non-limiting examples of ssRNA-RT viruses include retroviruses,alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus,epsilonretrovirus, lentivirus, spuma virus, metavirirus, andpseudoviruses. Non-limiting examples of dsDNA-RT viruses includehepadenovirus and caulimovirus. The virus can be a DNA virus. The viruscan be a RNA virus. The DNA virus may be a double-stranded DNA (dsDNA)virus. In some instances, the dsDNA virus is an adenovirus, herpesvirus, or pox virus. Examples of adenoviruses include, but are notlimited to, adenovirus and infectious canine hepatitis virus. Examplesof herpes viruses include, but are not limited to, herpes simplex virus,varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus. Anon-limiting list of pox viruses includes smallpox virus, cow pox virus,sheep pox virus, monkey pox virus, and vaccinia virus. The DNA virus maybe a single-stranded DNA (ssDNA) virus. The ssDNA virus may be aparvovirus. Examples of parvoviruses include, but are not limited to,parvovirus B19, canine parvovirus, mouse parvovirus, porcine parvovirus,feline panleukopenia, and Mink enteritis virus.

The virus can be a RNA virus. The RNA virus may be a double-stranded RNA(dsRNA) virus, (+) sense single-stranded RNA virus ((+)ssRNA) virus, or(−) sense single-stranded ((−) ssRNA) virus. A non-limiting list ofdsRNA viruses include reovirus, orthoreovirus, cypovirus, rotavirus,bluetongue virus, and phytoreovirus. Examples of (+) ssRNA virusesinclude, but are not limited to, picornavirus and togavirus. Examples ofpicornaviruses include, but are not limited to, enterovirus, rhinovirus,hepatovirus, cardiovirus, aphthovirus, poliovirus, parechovirus,erbovirus, kobuvirus, teschovirus, and coxsackie. In some instances, thetogavirus is a rubella virus, Sindbis virus, Eastern equine encephalitisvirus, Western equine encephalitis virus, Venezuelan equine encephalitisvirus, Ross River virus, O'nyong'nyong virus, Chikungunya, or SemlikiForest virus. A non-limiting list of (−) ssRNA viruses includeorthomyxovirus and rhabdovirus. Examples of orthomyxoviruses include,but are not limited to, influenzavirus a, influenzavirus B,influenzavirus C, isavirus, and thogotovirus. Examples of rhabdovirusesinclude, but are not limited to, cytorhabdovirus, dichorhabdovirus,ephemerovirus, lyssavirus, novirhabdovirus, and vesiculovirus.

Fetal Disorders

In some instances, the disease or condition is pregnancy. The methodsand kits disclosed herein may comprise diagnosing a fetal condition in apregnant subject. The methods and kits disclosed herein may compriseidentifying fetal mutations or genetic abnormalities. The molecules tobe stochastically labeled may be from a fetal cell or tissue.Alternatively, or additionally, the molecules to be labeled may be fromthe pregnant subject.

The methods and kits disclosed herein may be used in the diagnosis,prediction or monitoring of autosomal trisomies (e.g., Trisomy 13, 15,16, 18, 21, or 22). In some cases the trisomy may be associated with anincreased chance of miscarriage (e.g., Trisomy 15, 16, or 22). In othercases, the trisomy that is detected is a liveborn trisomy that mayindicate that an infant will be born with birth defects (e.g., Trisomy13 (Patau Syndrome), Trisomy 18 (Edwards Syndrome), and Trisomy 21 (DownSyndrome)). The abnormality may also be of a sex chromosome (e.g., XXY(Klinefelter's Syndrome), XYY (Jacobs Syndrome), or XXX (Trisomy X). Themolecule(s) to be labeled may be on one or more of the followingchromosomes: 13, 18, 21, X, or Y. For example, the molecule is onchromosome 21 and/or on chromosome 18, and/or on chromosome 13.

Further fetal conditions that may be determined based on the methods andkits disclosed herein include monosomy of one or more chromosomes (Xchromosome monosomy, also known as Turner's syndrome), trisomy of one ormore chromosomes (13, 18, 21, and X), tetrasomy and pentasomy of one ormore chromosomes (which in humans is most commonly observed in the sexchromosomes, e.g.,)(XXX, XXYY, XXXY, XYYY, XXXXY, XXXYY, XYYYY andXXYYY), monoploidy, triploidy (three of every chromosome, e.g., 69chromosomes in humans), tetraploidy (four of every chromosome, e.g., 92chromosomes in humans), pentaploidy and multiploidy.

Further disclosed herein is a method of forensic analysis comprising anyof the above described methods. Forensic scientists may use nucleicacids in various samples (e.g., blood, semen, skin, saliva, hair) foundat a crime scene to identify the presence of an individual at the scene,such as a perpetrator. This process is formally termed DNA profiling,but may also be called “genetic fingerprinting.” For example, DNAprofiling comprises measuring and comparing the lengths of variablesections of repetitive DNA, such as short tandem repeats andminisatellites, in various samples and people. This method is usually anextremely reliable technique for matching a DNA sample from a personwith DNA in a sample found at the crime scene. However, identificationmay be complicated if the scene is contaminated with DNA from severalpeople. In this instance, as well as in other forensic applications, itmay be advantageous to obtain absolute quantification of nucleic acidsfrom a single cell or small number of cells.

In some instances, the disease or condition is an immune disorder. Animmune diorder can be an inflammatory disorder, an autoimmune disorder,irritable bowel syndrome or ulcerative colitis. Examples of autoimmunediseases include Chrohn's disease, lupus, and Graves' disease.

In some instances, the disease or disorder is a neorlogical condition ordisorder. A neorlogical condition or disorder can be AcquiredEpileptiform Aphasia, Acute Disseminated Encephalomyelitis,Adrenoleukodystrophy, Agenesis of the corpus callosum, Agnosia, Aicardisyndrome, Alexander disease, Alpers' disease, Alternating hemiplegia,Alzheimer's disease, Amyotrophic lateral sclerosis (see Motor NeuronDisease), Anencephaly, Angelman syndrome, Angiomatosis, Anoxia, Aphasia,Apraxia, Arachnoid cysts, Arachnoiditis, Arnold-Chiari malformation,Arteriovenous malformation, Asperger's syndrome, Ataxia Telangiectasia,Attention Deficit Hyperactivity Disorder, Autism, Auditory processingdisorder, Autonomic Dysfunction, Pain, Batten disease, Behcet's disease,Bell's palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy,Benign Intracranial Hypertension, Bilateral frontoparietalpolymicrogyria, Binswanger's disease, Blepharospasm, Bloch-Sulzbergersyndrome, Brachial plexus injury, Brain abscess, Brain damage, Braininjury, Brain tumor, Brown-Sequard syndrome, Canavan disease, Carpaltunnel syndrome (CTS), Causalgia, Central pain syndrome, Central pontinemyelinolysis, Centronuclear myopathy, Cephalic disorder, Cerebralaneurysm, Cerebral arteriosclerosis, Cerebral atrophy, Cerebralgigantism, Cerebral palsy, Charcot-Marie-Tooth disease, Chiarimalformation, Chorea, Chronic inflammatory demyelinating polyneuropathy(CIDP), Chronic pain, Chronic regional pain syndrome, Coffin Lowrysyndrome, Coma, including Persistent Vegetative State, Complex Ideficiency syndrome, Complex I deficiency syndrome, Complex IIdeficiency syndrome, Complex III deficiency syndrome, Complex IV/COXdeficiency syndrome, Complex V deficiency syndrome, Congenital facialdiplegia, Corticobasal degeneration, Cranial arteritis,Craniosynostosis, Creutzfeldt-Jakob disease, Cumulative traumadisorders, Cushing's syndrome, Cytomegalic inclusion body disease (CMD),Cytomegalovirus Infection, Dandy-Walker syndrome, Dawson disease,Deficiency of mitochondrial NADH dehydrogenase component of Complex I,De Morsier's syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease,Delayed sleep phase syndrome, Dementia, Dermatomyositis, NeurologicalDyspraxia, Diabetic neuropathy, Diffuse sclerosis, Dysautonomia,Dyscalculia, Dysgraphia, Dyslexia, Dystonia, Early infantile epilepticencephalopathy, Empty sella syndrome, Encephalitis, Encephalocele,Encephalotrigeminal angiomatosis, Encopresis, Epilepsy, Erb's palsy,Erythromelalgia, Essential tremor, Fabry's disease, Fahr's syndrome,Fainting, Familial spastic paralysis, Febrile seizures, Fisher syndrome,Friedreich's ataxia, FART Syndrome, Gaucher's disease, Gerstmann'ssyndrome, Giant cell arteritis, Giant cell inclusion disease, Globoidcell Leukodystrophy, Gray matter heterotopia, Guillain-Barre syndrome,HTLV-1 associated myelopathy, Hallervorden-Spatz disease, Head injury,Headache, Hemifacial Spasm, Hereditary Spastic Paraplegia, Heredopathiaatactica polyneuritiformis, Herpes zoster oticus, Herpes zoster,Hirayama syndrome, Holoprosencephaly, Huntington's disease,Hydranencephaly, Hydrocephalus, Hypercortisolism, Hypoxia,Immune-Mediated encephalomyelitis, Inclusion body myositis,Incontinentia pigmenti, Infantile phytanic acid storage disease,Infantile Refsum disease, Infantile spasms, Inflammatory myopathy,Intracranial cyst, Intracranial hypertension, Joubert syndrome,Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, KlippelFeil syndrome, Krabbe disease, Kufor-Rakeb syndrome, Kugelberg-Welanderdisease, Kuru, Lafora disease, Lambert-Eaton myasthenic syndrome,Landau-Kleffner syndrome, Lateral medullary (Wallenberg) syndrome,Learning disabilities, Leigh's disease, Lennox-Gastaut syndrome,Lesch-Nyhan syndrome, Leukodystrophy, Lewy body dementia, Lissencephaly,Locked-In syndrome, Lou Gehrig's disease, Lumbar disc disease, Lymedisease-Neurological Sequelae, Machado-Joseph disease (Spinocerebellarataxia type 3), Macrencephaly, Maple Syrup Urine Disease,Megalencephaly, Melkersson-Rosenthal syndrome, Menieres disease,Meningitis, Menkes disease, Metachromatic leukodystrophy, Microcephaly,Migraine, Miller Fisher syndrome, Mini-Strokes, Mitochondrial disease,Mitochondrial dysfunction, Mitochondrial Myopathies, MitochondrialRespiratory Chain Complex I Deficiency, Mobius syndrome, Monomelicamyotrophy, Motor Neuron Disease, Motor skills disorder, Moyamoyadisease, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal motorneuropathy, Multiple sclerosis, Multiple system atrophy with posturalhypotension, Muscular dystrophy, Myalgic encephalomyelitis, Myastheniagravis, Myelinoclastic diffuse sclerosis, Myoclonic Encephalopathy ofinfants, Myoclonus, Myopathy, Myotubular myopathy, Myotonia congenita,NADH-coenzyme Q reductase deficiency, NADH:Q(1) oxidoreductasedeficiency, Narcolepsy, Neurofibromatosi s, Neuroleptic malignantsyndrome, Neurological manifestations of AIDS, Neurological sequelae oflupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migrationdisorders, Niemann-Pick disease, Non 24-hour sleep-wake syndrome,Nonverbal learning disorder, O'Sullivan-McLeod syndrome, OccipitalNeuralgia, Occult Spinal Dysraphism Sequence, Ohtahara syndrome,Olivopontocerebellar atrophy, Opsoclonus myoclonus syndrome, Opticneuritis, Orthostatic Hypotension, Overuse syndrome, oxidativephosphorylation disorders, Palinopsia, Paresthesia, Parkinson's disease,Paramyotonia Congenita, Paraneoplastic diseases, Paroxysmal attacks,Parry-Romberg syndrome (also known as Rombergs Syndrome),Pelizaeus-Merzbacher disease, Periodic Paralyses, Peripheral neuropathy,Persistent Vegetative State, Pervasive neurological disorders, Photicsneeze reflex, Phytanic Acid Storage disease, Pick's disease, PinchedNerve, Pituitary Tumors, PMG, Polio, Polymicrogyria, Polymyositis,Porencephaly, Post-Polio syndrome, Postherpetic Neuralgia (PHN),Postinfectious Encephalomyelitis, Postural Hypotension, Prader-Willisyndrome, Primary Lateral Sclerosis, Prion diseases, ProgressiveHemifacial Atrophy also known as Rombergs Syndrome, Progressivemultifocal leukoencephalopathy, Progressive Sclerosing Poliodystrophy,Progressive Supranuclear Palsy, Pseudotumor cerebri, Ramsay-Huntsyndrome (Type I and Type II), Rasmussen's encephalitis, Reflexsympathetic dystrophy syndrome, Refsum disease, Repetitive motiondisorders, Repetitive stress injury, Restless legs syndrome,Retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome,Rombergs Syndrome, Rabies, Saint Vitus dance, Sandhoff disease,Schytsophrenia, Schilder's disease, Schizencephaly, Sensory IntegrationDysfunction, Septo-optic dysplasia, Shaken baby syndrome, Shingles,Shy-Drager syndrome, Sjogren's syndrome, Sleep apnea, Sleeping sickness,Snatiation, Sotos syndrome, Spasticity, Spina bifida, Spinal cordinjury, Spinal cord tumors, Spinal muscular atrophy, Spinal stenosis,Steele-Richardson-Olszewski syndrome, see Progressive SupranuclearPalsy, Spinocerebellar ataxia, Stiff-person syndrome, Stroke,Sturge-Weber syndrome, Subacute sclerosing panencephalitis, Subcorticalarteriosclerotic encephalopathy, Superficial siderosis, Sydenham'schorea, Syncope, Synesthesia, Syringomyelia, Tardive dyskinesia,Tay-Sachs disease, Temporal arteritis, Tethered spinal cord syndrome,Thomsen disease, Thoracic outlet syndrome, Tic Douloureux, Todd'sparalysis, Tourette syndrome, Transient ischemic attack, Transmissiblespongiform encephalopathies, Transverse myelitis, Traumatic braininjury, Tremor, Trigeminal neuralgia, Tropical spastic paraparesis,Trypanosomiasis, Tuberous sclerosis, Vasculitis including temporalarteritis, Von Hippel-Lindau disease (VHL), Viliuisk Encephalomyelitis(VE), Wallenberg's syndrome, Werdnig-Hoffman disease, West syndrome,Whiplash, Williams syndrome, Wilson's disease, X-Linked Spinal andBulbar Muscular Atrophy, or Zellweger syndrome.

DEFINITIONS

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

As used herein, the term “label” may refer to a unique oligonucleotidesequence that may allow a corresponding nucleic acid base and/or nucleicacid sequence to be identified. In some embodiments, the nucleic acidbase and/or nucleic acid sequence may be located at a specific positionon a larger polynucleotide sequence (e.g., a polynucleotide attached toa bead).

As used herein, the term “hybridization” may refer to the process inwhich two single-stranded polynucleotides bind non-covalently to form astable double-stranded polynucleotide. The term “hybridization” may alsorefer to triple-stranded hybridization. The resulting (usually)double-stranded polynucleotide is a “hybrid” or “duplex.”

As used herein, “nucleoside” may include natural nucleosides, such as2′-deoxy and 2′-hydroxyl forms. “Analogs” in reference to nucleosidesmay include synthetic nucleosides comprising modified base moietiesand/or modified sugar moieties, or the like. Analogs may be capable ofhybridization. Analogs may include synthetic nucleosides designed toenhance binding properties, reduce complexity, increase specificity, andthe like. Exemplary types of analogs may include oligonucleotidephosphoramidates (referred to herein as “amidates”), peptide nucleicacids (referred to herein as “PNAs”), oligo-2′-O-alkylribonucleotides,polynucleotides containing C-5 propynylpyrimidines, and locked nucleicacids (LNAs).

As used herein, the terms “nucleic acid molecule,” “nucleic acidsequence,” “nucleic acid fragment,” “oligonucleotide,” “oligonucleotidefragment” and “polynucleotide” may be used interchangeably and may beintended to include, but are not limited to, polymeric forms ofnucleotides that may have various lengths, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Nucleic acid molecules mayinclude single stranded DNA (ssDNA), double stranded DNA (dsDNA), singlestranded RNA (ssRNA) and double stranded RNA (dsRNA). Different nucleicacid molecules may have different three-dimensional structures, and mayperform various functions. Non-limiting examples of nucleic acidmolecules may include a gene, a gene fragment, a genomic gap, an exon,an intron, intergenic DNA (including, without limitation,heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, small interfering RNA (siRNA), miRNA, small nucleolar RNA(snoRNA), cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of a sequence, isolated RNA of asequence, nucleic acid probes, and primers.

Oligonucleotides may refer to a linear polymer of natural or modifiednucleosidic monomers linked by phosphodiester bonds or analogs thereof.An “oligonucleotide fragment” refers to an oligonucleotide sequence thathas been cleaved into two or more smaller oligonucleotide sequences.Oligonucleotides may be natural or synthetic. Oligonucleotides mayinclude deoxyribonucleosides, ribonucleosides, and non-natural analogsthereof, such as anomeric forms thereof, peptide nucleic acids (PNAs),and the like. Oligonucleotides may be capable of specifically binding toa target genome by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like.Oligonucleotides and the term “polynucleotides” may be usedinterchangeably herein.

Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGCCTG,” it may be understood that the nucleotides are in 5′to 3′ order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotesdeoxythymidine, and “U” denotes the ribonucleoside, uridine, unlessotherwise noted.

Oligonucleotides may include one or more non-standard nucleotide(s),nucleotide analog(s) and/or modified nucleotides. Examples of modifiednucleotides may include, but are not limited to diaminopurine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,oligonucleotide phosphoramidates (referred to herein as “amidates”),peptide nucleic acids (referred to herein as “PNAs”),oligo-2′-O-alkylribonucleotides, polynucleotides containing C-5propynylpyrimidines, locked nucleic acids (LNAs), 2,6-diaminopurine andthe like. Nucleic acid molecules may also be modified at the base moiety(e.g., at one or more atoms that typically are available to form ahydrogen bond with a complementary nucleotide and/or at one or moreatoms that are not typically capable of forming a hydrogen bond with acomplementary nucleotide), sugar moiety or phosphate backbone.

As used herein, a “sample” may refer to a single cell or many cells.Nucleic acid molecules may be obtained from one or more samples. Asample may comprise a single cell type or a combination of two or morecell types. A sample may include a collection of cells that perform asimilar function such as those found, for example, in a tissue. A samplemay comprise one or more tissues. Examples of tissues may include, butare not limited to, epithelial tissue (e.g., skin, the lining of glands,bowel, skin and organs such as the liver, lung, kidney), endothelium(e.g., the lining of blood and lymphatic vessels), mesothelium (e.g.,the lining of pleural, peritoneal and pericardial spaces), mesenchyme(e.g., cells filling the spaces between the organs, including fat,muscle, bone, cartilage and tendon cells), blood cells (e.g.,erythrocytes, granulocytes, neutrophils, eosinophils, basophils,monocytes, T-lymphocytes (also known as T-cells), B-lymphocytes (alsoknown as B-cells), plasma cells, megakaryocytes and the like), neurons,germ cells (e.g., spermatozoa, oocytes), amniotic fluid cells, placenta,stem cells and the like. A sample may be obtained from one or more ofsingle cells in culture, metagenomic samples, embryonic stem cells,induced pluripotent stem cells, cancer samples, tissue sections, andbiopsies, or any combination thereof.

As used herein, the term “organism” may include, but is not limited to,a human, a non-human primate, a cow, a horse, a sheep, a goat, a pig, adog, a cat, a rabbit, a mouse, a rat, a gerbil, a frog, a toad, a fish(e.g., Danio rerio) a roundworm (e.g., C. elegans) and any transgenicspecies thereof. The term “organism” may also include, but is notlimited to, a yeast (e.g., S. cerevisiae) cell, a yeast tetrad, a yeastcolony, a bacterium, a bacterial colony, a virion, virosome, virus-likeparticle and/or cultures thereof, and the like.

As used herein, the term “attach,” “conjugate,” and “couple” may be usedinterchangeably and may refer to both covalent interactions andnoncovalent interactions.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Enzymatic Split-Pool Synthesis

In this example, an enzymatic split-pool synthesis method was used toproduce oligonucleotide coupled beads. As shown in FIG. 2A, a set ofoligonucleotides was added to each well of a first plate. Anoligonucleotide in a set of oligonucleotides comprises a 5′ amine,universal sequence, cell label and a linker. The 5′ amine, universalsequence and linker are the same for each set of oligonucleotides. Theuniversal sequence and linker are different from each other. However,the cell label is different for each set of oligonucleotides. Thus, eachwell has a different cell label. In Step 1 of the enzymatic split-poolsynthesis, oligonucleotide-coupled beads were synthesized by adding asingle bead to each well and performing1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling reactions.The oligonucleotides beads resulting from Step 1 comprise a bead coupledto multiple oligonucleotides. The oligonucleotide comprises a 5′-amine,universal sequence, cellular label 1, and linker 1 (see FIG. 2A). Theoligonucleotides on the same bead are the same. However,oligonucleotides on a first bead are different from oligonucleotides ona second bead.

In Step 2 of the enzymatic split-pool synthesis, multiple washes wereperformed to remove uncoupled oligonucleotides. Once the uncoupledoligonucleotides were removed, the oligonucleotide-coupled beads werepooled (see FIG. 2A). The oligonucleotide coupled beads resulting fromStep 2 comprise a bead coupled to multiple single strandedoligonucleotides. The single stranded oligonucleotide comprises a 5′amine, universal sequence, cell label 1 and linker 1. Eacholigonucleotide on a bead is identical. However, each bead comprises adifferent oligonucleotide. The oligonucleotides coupled to the differentbeads differ by the cell label 1 sequence.

As shown in FIG. 2B, a set of oligonucleotides was added to each well ofa second plate. An oligonucleotide in a set of oligonucleotidescomprises a first linker, cell label, and a second linker. The first andsecond linkers are the same for each set of oligonucleotides. The firstand second linkers are different from each other. However, the celllabel is different for each set of oligonucleotides. Thus, each well hasa different cell label.

In Step 3 of the enzymatic split-pool synthesis, the oligonucleotidecoupled beads that were pooled in Step 2 were split into the wells ofthe second plate. Because the first linker of the oligonucleotides inthe wells of the second plate are complementary to the linker of theoligonucleotides coupled to the beads, primer extension using Klenowlarge fragment was performed to couple the oligonucleotides from thesecond plate to the oligonucleotide coupled beads from Step 2. Theoligonucleotides coupled beads resulting from Step 3 comprise a beadcoupled to multiple double stranded oligonucleotides. The doublestranded oligonucleotide comprises a 5′ amine, universal sequence, celllabel 1, linker 1, cell label 2, and linker 2 (see FIG. 2B).

In Step 4 of the enzymatic split-pool synthesis, multiple washes wereperformed to remove uncoupled oligonucleotides and the Klenow largefragment enzymes. The second plate was heated to denature the doublestranded oligonucleotides, and the oligonucleotide coupled beads werepooled (see FIG. 2B). The oligonucleotide coupled beads resulting fromStep 4 comprise a bead coupled to multiple single strandedoligonucleotides. The single stranded oligonucleotide comprises a 5′amine, universal sequence, cell label 1, linker 1, cell label 2, andlinker 2. Each oligonucleotide on a bead is identical. However, eachbead comprises a different oligonucleotide. The oligonucleotides coupledto the different beads differ by the combined cell label sequences. Forexample, a first bead may comprise oligonucleotides comprising a firstcell label of cell label A and second cell label of cell label C and asecond bead may comprise oligonucleotides comprising a first cell labelof cell label C and a second cell label of cell label D. Thus, the firstbead and the second bead may comprise the same cell label (in this case,cell label C), however, the combined cell label sequences of the firstbead and the second bead are different (e.g., for the first bead, thecombined cell label sequence is cell label A+ cell label C; for thesecond bead, the combined cell label sequence is cell label C+ celllabel A). In other instances, two beads may comprise oligonucleotidescomprising different cell labels. For example, a first bead may compriseoligonucleotides comprising cell label A and cell label B and a secondbead may comprise oligonucleotides comprising cell label C and celllabel D. In this instance, both of the cell labels of the first bead aredifferent from both of the cell labels of the second bead.

As shown in FIG. 2C, a set of oligonucleotides was added to each well ofa third plate. An oligonucleotide in a set of oligonucleotides comprisesa linker, cell label, molecular label, and an oligodT. The linker andoligodT sequences are the same for each set of oligonucleotides.However, the cell label is different for each set of oligonucleotides.Thus, each well has a different cell label. In addition, the molecularlabel is different for oligonucleotides within a set. Thus, a singlewell contains a plurality of oligonucleotides with the same cell label,but different molecular labels. The oligonucleotides from differentwells may contain the same molecular label.

In Step 5 of the enzymatic split-pool synthesis, the oligonucleotidecoupled beads that were pooled in Step 4 were split into the wells ofthe third plate. Because the linker of the oligonucleotides in the wellsof the second plate are complementary to the second linker of theoligonucleotides coupled to the beads, primer extension using Klenowlarge fragment was performed to couple the oligonucleotides from thethird plate to the oligonucleotide coupled beads from Step 4. Theoligonucleotides coupled beads resulting from Step 5 comprise a beadcoupled to multiple double stranded oligonucleotides. The doublestranded oligonucleotide comprises a 5′ amine, universal sequence, celllabel 1, linker 1, cell label 2, linker 2, cell label 3, molecular labeland oligodT (see FIG. 2C).

In Step 6 of the enzymatic split-pool synthesis, multiple washes wereperformed to remove uncoupled oligonucleotides and the Klenow largefragment enzymes. The third plate was heated to denature the doublestranded oligonucleotides, and the oligonucleotide coupled beads werepooled (see FIG. 2C). The oligonucleotide coupled beads resulting fromStep 4 comprise a bead coupled to multiple single strandedoligonucleotides. The single stranded oligonucleotide comprises a 5′amine, universal sequence, cell label 1, linker 1, cell label 2, linker2, cell label 3, molecular label and oligodT. The multiple singlestranded oligonucleotides on a single bead may be differentiated by themolecular label. The cell label portions of the multipleoligonucleotides on a single bead are identical. Each bead comprisesdifferent oligonucleotides. The oligonucleotides coupled to thedifferent beads differ by the cell label sequences. The molecular labelon the oligonucleotides from different beads may be the same. Themolecular label on the oligonucleotides from different beads may bedifferent. Two or more beads may differ by the combined cell labelsequences. For example, a first bead may comprise an oligonucleotidecomprising cell label A, cell label B and cell label C and a second beadmay comprise an oligonucleotide comprising cell label B, cell label Dand cell label A. In this instance, the first and second bead bothcontain cell label B, however the two other cell labels are different.Thus, two or more beads may comprise oligonucleotides differing by atleast one cell label. Two or more beads may comprise oligonucleotidesdiffering by at least two cell labels. Two or more beads may compriseoligonucleotides differing by at least three cell labels. However, abead may comprise an oligonucleotide comprising two or more identicalcell labels. For example, a bead may comprise an oligonucleotidecomprising cell label A, cell label A and cell label D. A bead maycomprise oligonucleotides comprising at least three identical celllabels. For example, a bead may comprise an oligonucleotide comprisingcell label A, cell label A and cell label A. A bead may compriseoligonucleotides comprising three non-identical cell labels. Forexample, a bead may comprise an oligonucleotide comprising cell label A,cell label D and cell label E. A bead may comprise at least twooligonucleotides comprising at least two different molecular labels. Forexample, a bead may comprise a first oligonucleotide comprisingmolecular label A and a second oligonucleotide comprising molecularlabel D. However, a bead may comprise multiple copies of anoligonucleotide comprising a first molecular label. Thus, a bead maycomprise at least two oligonucleotides comprising the same molecularlabel. For example, a bead may comprise a first oligonucleotidecomprising molecular label A and a second oligonucleotide comprisingmolecular label A. At least 30% of the oligonucleotides on a bead maycomprise different molecular labels. At least 40% of theoligonucleotides on a bead may comprise different molecular labels. Atleast 50% of the oligonucleotides on a bead may comprise differentmolecular labels. At least 60% of the oligonucleotides on a bead maycomprise different molecular labels. Less than 30% of theoligonucleotides on a bead may comprise the same molecular label. Lessthan 20% of the oligonucleotides on a bead may comprise the samemolecular label. Less than 15% of the oligonucleotides on a bead maycomprise the same molecular label. Less than 10% of the oligonucleotideson a bead may comprise the same molecular label. Less than 5% of theoligonucleotides on a bead may comprise the same molecular label.

The enzymatic split-pool synthesis technique may be performed onmultiple plates or plates with a greater number of wells to produce alarger number of oligonucleotide coupled beads. The use of threeseparate cell label portions may increase the diversity of the totalcell label portions on the beads. With 96 different sequence options foreach cell label portion, 884,736 different cell label combinations maybe created.

Example 2 Comparison of Amplification in Tube and Microwell

The disclosure provides a method for capturing cells. About 5,000 Ramoscells were captured on a microwell array comprising microwells of about30 micron in diameter. Some cells were not captured. The control for theexperiment was an equivalent number of cells captured in a tube. Boththe cells in the tube and the cells in the microwell array were lysed.The nucleic acid was allowed to hybridize to a conjugated bead. Realtime PCR of GAPDH and RPL19 genes was performed.

FIG. 9 shows the results of the real time PCR amplification. The yieldfrom the microwell was larger than the yield from the nucleic acid inthe tube, indicating that the hybridization of the nucleic acid to theoligonucleotide was more effective in the microwell than the tube(compare grey bar and white bar, respectively).

Example 3 Comparison of Amplification of Second Synthesized Strand andSynthesis on Bead

Cells were obtained and lysed as described in Example 1. RPL19, TUBB,and GAPDH were amplified either off the second strand synthesized offthe solid supports, or direct on the solid supports using a universalprimer. FIG. 10 shows, amplification directly on the solid supports(FIG. 10) yielded less off-target amplification than amplification notdirectly off a solid support. GAPDH and TUBB amplifications producedcorrectly sized products regardless of method (the left lane of eachtriplet in FIG. 10 corresponds to solid support plus lysate in tubeformat, the middle lane of each triplet corresponds to solid supportsfrom the microwell, and the right lane of each triplet corresponds tosolid supports plus purified nucleic acid). The RPL19 product hadminimal off-target amplification products, but only produced a strongproduct when purified nucleic acid was used with the solid support.These experiments indicate that amplification directly on the beadsproduces less off-target amplification products than amplification usinga second strand synthesized off the solid support.

Example 4 Multiplex Analysis of Target Nucleic Acids

Cells are obtained and lysed as described in Example 1. Target nucleicacids are hybridized to the solid support comprising oligonucleotides. Aplurality of copies of the target nucleic acid are hybridized to atarget binding region comprising an oligodT sequence. The plurality ofcopies of the target nucleic acid are reverse transcribed using reversetranscriptase. Reverse transcription incorporates the features of theoligonucleotide to which the copy of the target nucleic acid washybridized (e.g., the molecular label, the cellular label, and theuniversal label). The plurality of copies of the target nucleic acid areamplified using PCR. The amplified copies of the target nucleic acid aresequenced. The sequenced target nucleic acids are counted to determinethe copy number of the target nucleic acid in the cell. The counting isperformed by counting the number of different molecular labels for eachof the same sequence read of target nucleic acid. In this way,amplification bias may be diminished.

Example 5 Evaluating Efficacy of Split-Pool Synthesis to Produce Beadswith Clonal Copies of One Cell Label Combination

In this example, the efficacy of split-pool synthesis to produce beadswith clonal copies of one cell label combination was evaluated.Oligonucleotide coupled beads were synthesized by the enzymaticsplit-pool synthesis method as described in Example 1. 250 ng of totalRNA was purified from Ramos cells, which is equivalent to RNA from25,000 cells. The total RNA was contacted with 35,000 oligonucleotidecoupled beads, resulting in hybridization of mRNA to the oligonucleotidecoupled beads. cDNA synthesis was performed on the mRNA hybridized tothe oligonucleotide coupled beads. Samples comprising 18, 175, and 1750beads were used for further analysis. PCR amplification reactions usingGAPDH-specific primers and IGJ-specific primers were performed on thecDNA bound to the beads from the 18-, 175- and 1750-bead samples. ThecDNA molecules attached to the beads were sequenced. FIG. 11A-I showgraphical representations of the sequencing results. For FIG. 11A-C, thenumber of reads per bead is plotted on the y-axis and the unique barcode(e.g., cell label combination) is plotted on the x-axis for the 18-bead,175-bead and 1750-bead samples, respectively. For FIG. 11D-F, the numberof unique molecules per bead is plotted on the y-axis and the uniquebarcode (e.g., cell label combination) is plotted on the x-axis for the18-bead, 175-bead and 1750-bead samples, respectively. For FIG. 11G-I,the number of unique molecules per bead is plotted on the y-axis and theunique barcode is plotted on the x-axis for the 18-bead, 175-bead and1750-bead samples, respectively. The results for FIG. 11G-I are sortedby the total number of molecules. The median number of unique moleculesper bead for the various samples is shown in Table 1. Numerical valuesfor the sequencing results are shown in Table 2. For FIG. 11J-L, thenumber of unique barcode (bc) combination using the index is plotted onthe y-axis and the barcode (bc) segment index is plotted on the x-axisfor the cell label 1, cell label 2, and cell label 3 for the 1750-beadsample, respectively. The barcode (bc) refers to the cell label (e.g.,bc segment1=cell label part 1). As shown in FIG. 11J-L, the presence ofalmost all 96 barcodes within each segment was detected by sequencing.These results demonstrate the success of the enzymatic split-poolsynthesis method to produce beads with clonal copies of one cell labelcombination.

TABLE 1 Median number of unique molecules 18 beads 175 beads 1750 beadsIGJ 78 85 40 GAPDH 22 45 25

TABLE 2 Expected # of beads 17.5 175 1750 Total number of reads 5832160308 133043 >=8 match in constant 1 56385 57615 123349 >=8 match inconstant 2 54117 55187 115126 >=8 match in constant 1 & 2 54114 55185115107 Perfect match in all 3 sub-barcodes 38585 46066 95217 Perfectmatch in gene (40 bp) 29968 33775 72260 Total number of unique barcode239 407 1654 combination % useful reads 51.388% 56.00% 54.31% Number ofunique barcode 5 26 288 combinations >20 read

Example 6 Single Cell RNA Labeling Using Oligonucleotide Coupled Beads

In this example, the efficacy of single cell RNA labeling usingoligonucleotide coupled beads was evaluated. Three cell samples wereprepared as follows:

Sample 1: Sample 2: Sample 3: K562 Ramos Ramos + only only K562 mixtureNumber of microwells ~10000 ~10000 ~10000 Number of Ramos cells 0 50003750 Number of K562 cells 1000 0 2500

The cell suspension of the samples was added to the top of a microwelland cells were allowed to settle into the wells of the microwell array.Cells not captured by the microwell array were washed away in aphosphate buffered saline (PBS) bath. Oligonucleotide coupled beads, asprepared by the enzymatic split-pool synthesis method described inExample 1, were added to the microwell array. The oligonucleotidecoupled bead comprises a magnetic bead with a plurality ofoligonucleotides. Each oligonucleotide on the bead comprises a 5′ amine,universal sequence, cell label 1, linker 1, cell label 2, linker 2, celllabel 3, molecular label, and oligodT. For each oligonucleotide on thesame bead, the sequences of the oligonucleotides are identical exceptfor the molecular label. For oligonucleotides on different beads, thecell label 1, 2, and 3 combinations are different. Approximately 5-6beads were added per well of the microwell array. In some instances, forevery 10 wells, 50 beads may be deposited on the array, with 0-2 beadsfalling into each well. The beads were allowed to settle into the wellsand uncaptured beads were washed away in a PBS bath. A magnet was placedunderneath the microwell array. Cells were lysed by the addition of coldlysis buffer. The array and magnet were placed on a cold aluminum blockfor 5 minutes. mRNA from the lysed cells were hybridized to theoligonucleotides coupled to the beads. The array was washed with excesslysis buffer to remove unbound mRNA. The beads were retrieved from thewells by placing a magnet on top of the microwell array. The retrievedbeads were washed. cDNA synthesis was performed on the beads usingSuperscript III at 50° C. for 50 minutes on a rotor. Non-extendedoligodT from the oligonucleotides on the beads were removed by ExoItreatment conducted at 37° C. for 30 minutes on a rotor. Gene-specificPCR amplification was conducted on the cDNA. The genes selected for thegene-specific PCR were cell-type specific and are shown in Table 3. ThePCR amplified products were sequenced. Sequencing statistics are shownin Table 4. FIGS. 12A-C show a histogram of the sequencing results forthe K562-only sample, Ramos-only sample, and K562+Ramos mixture sample,respectively. For FIG. 12A-C, the unique molecule per barcode plotted onthe y-axis and the unique bc combination index, sorted by read per bcplotted on the x-axis.

TABLE 3 Number Gene Cell-type  1 CD74 Ramos-specific  2 CD79aRamos-specific  3 IGJ Ramos-specific  4 TCL1A Ramos-specific  5 SEPT9Ramos-specific  6 CD27 Ramos-specific  7 CD41 K562-specific  8 GYPAK562-specific  9 GATA1 K562-specific 10 GATA2 K562-specific 11 HBG1K562-specific 12 GAPDH common

TABLE 4 Sample 1: Sample 2: Sample 3: K562 Ramos Ramos + only only K562mixture Number of Ramos cells 0 5000 3750 Number of K562 cells 1000 02500 Total number of reads 717718 1329189 2399025 >=8 match in constant1 657911 1201081 2026726 >=8 match in constant 2 581581 10713641513466 >=8 match in constant 1 & 2 581508 1071153 1513102 Perfect matchin all 3 481564 862348 1248073 sub-barcodes Perfect match in gene (40bp) 283463 575713 1004338 % useful reads 39.50% 43.31% 41.86% Totalnumber of unique 8501 29647 28783 barcode combination Number of unique145 1072 768 barcode combinations >30 molecule Capture efficiency 0.1450.2144 0.12288

Single cell labeling was used to determine the copy number for thesingle-cell type samples (e.g., K562-only sample, Ramos-only sample).FIG. 12D-E shows a graph of the copy number for genes listed in Table 3for the Ramos-only cell sample and K562-only cell sample, respectively.For FIG. 12D-E, the number of molecules per barcode (bc) combination isplotted on the y-axis and the unique barcode combination, sorted bytotal number of molecules per bc combination is plotted on the x-axis.The results shown in FIGS. 12D-E were based on sequencing data frombeads with >30 total number of unique molecules. These resultsdemonstrate that the proportion of molecules per amplicon per beadmatches expectations for the cell type. For the K562-only cell sample,the skew of the number of molecules is more severe and it appears thatHBG1, which is highly abundant in this cell type, has a variable copynumber. However, GAPDH copy number appears to be constant even thoughthe total number of molecules per bead is skewed. The copy number forthe individual genes are shown in FIG. 12F-I. For FIG. 12F-G, the copynumber is represented as copy per bead or single cell for Ramos-onlycells and K562-only cells, respectively. For FIG. 12H-I, the copy numberis represented as relative abundance per bead or single cell forRamos-only cells and K562-only cells, respectively.

Single cell labeling was used to determine the cell type of single cellsin the K562+Ramos mixture sample. Sequencing results from 100 uniquebarcode combinations with the most abundant molecules were analyzed toevaluate the efficacy of single cell labeling to determine the cell typeof single cells in the K562+Ramos mixture sample. FIG. 12J-M show graphsof the number of unique molecules per gene (y-axis) for the beads withthe 100 unique barcode combinations. The numbers on the x-axis refer tothe gene (see Table 3). FIG. 12J-M clearly depict general geneexpression patterns for the K562 and Ramos cells. FIG. 12N-O showenlarged graphs of two beads that depict the general pattern of geneexpression profiles for the two cell types. FIG. 12N shows the generalpattern of gene expression profile for K562-like cells and FIG. 12Oshows the general pattern of gene expression profile for Ramos-likecells. FIG. 12P shows a scatter plot of results based on principalcomponent analysis of gene expression profile of 768 beads with >30molecules per bead from the K562+Ramos mixture sample. Component 1,which is plotted on the x-axis, separates the two cell types. Component2, which is plotted on the y-axis, separates K562 cells with high andlow HBG1 copy number. Each dot on the scatter plot represents one uniquebarcode combination, which is equivalent to one bead or one cell. Basedon the principal component analysis, 409 beads corresponded to K562cells and 347 beads corresponded to Ramos cells. The copy number of thegenes from Table 3 was determined for the K562-like and Ramos-like celltypes. FIG. 12Q-R show histograms of the copy number per amplicon perbead for the K562-like cells (beads on the left of the first principalcomponent based on FIG. 12P) and Ramos-like cells (beads on the right ofthe first principal component based on FIG. 12P), respectively. For FIG.12Q-R, number of per bc combination is on the y-axis and unique barcodecombination, sorted by total number of molecules per bc combination ison the x-axis. FIG. 12S-T show the copy number per bead or single cellof the individual genes for the K562-like cells (beads on the left ofthe first principal component based on FIG. 12P) and Ramos-like cells(beads on the right of the first principal component based on FIG. 12P),respectively. Table 5 shows the mean copy number per bead for the singlecell and mixture samples.

TABLE 5 Single cell K562 + Ramos type samples mixture sample GeneK562-only Ramos-only K562-like Ramos-like CD74 0.00 39.95 0.10 7.50CD79a 0.02 30.97 0.84 18.88 IGJ 0.03 42.43 0.81 27.76 TCL1A 0.01 31.780.71 19.44 SEPT9 0.88 3.89 1.35 1.52 CD27 0.00 5.31 0.03 1.30 CD41 0.610.00 0.47 0.01 GYPA 1.92 0.00 0.73 0.02 GATA2 1.38 0.00 0.60 0.04 GATA10.94 0.00 1.04 0.04 HBG1 201.09 0.00 72.27 1.37 GAPDH 51.77 39.13 44.9413.53 GAPDH read 2.04 1.47 7.67 7.22 redundancy

Example 7 Evaluating Cross-Talk Between Beads

In this example, the cross-talk between beads was evaluated. Samplescomprising mixtures of mouse EL4 cells and Ramos cells were prepared asfollows:

High density Low density Number of microwells ~10000 ~10000 Number ofmouse EL4 cells 2500 1500 Number of Ramos cells 3750 1500

The cell suspension of the samples was added to the top of a microwelland cells were allowed to settle into the wells of the microwell array.Cells not captured by the microwell array were washed away in aphosphate buffered saline (PBS) bath. Oligonucleotide coupled beads, asprepared by the enzymatic split-pool synthesis method described inExample 1, were added to the microwell array. The oligonucleotidecoupled bead comprises a magnetic bead with a plurality ofoligonucleotides. Each oligonucleotide on the bead comprises a 5′ amine,universal sequence, cell label 1, linker 1, cell label 2, linker 2, celllabel 3, molecular label, and oligodT. For each oligonucleotide on thesame bead, the sequences of the oligonucleotides are identical exceptfor the molecular label. For oligonucleotides on different beads, thecell label 1, 2, and 3 combinations are different. Approximately 5-6beads were added per well of the microwell array. The beads were allowedto settle into the wells and uncaptured beads were washed away in a PBSbath. A magnet was placed underneath the microwell array. Cells werelysed by the addition of cold lysis buffer. The array and magnet wereplaced on a cold aluminum block for 5 minutes. mRNA from the lysed cellswere hybridized to the oligonucleotides coupled to the beads. The arraywas washed with excess lysis buffer to remove unbound mRNA. The beadswere retrieved from the wells by placing a magnet on top of themicrowell array. The retrieved beads were washed. cDNA synthesis wasperformed on the beads using Superscript III at 50° C. for 50 minutes ona rotor. Non-extended oligodT from the oligonucleotides on the beadswere removed by ExoI treatment conducted at 37° C. for 30 minutes on arotor. Gene-specific PCR amplification was conducted on the cDNA. Thegenes selected for the gene-specific PCR were cell-type specific and areshown in Table 6.

TABLE 6 Number Gene Cell-type 1 HS_CD74 human 2 HS_CD79a human 3 HS_IGJhuman 4 HS_TCL1A human 5 HS_SEPT9 human 6 HS_CD27 human 7 HS_GAPDH human8 MM_B2M mouse 9 MM_ACTM mouse 10 MM_HPRT mouse 11 MM_SHDA mouse

The PCR amplified products were sequenced. Sequencing statistics areshown in Table 7.

TABLE 7 Low density High density Number of Ramos cells 15000 3750 Numberof mouse cells 1500 2500 Total number of reads 2391780 4038217 >=8 matchin constant 1 2162945 3651643 >=8 match in constant 2 19818353356493 >=8 match in constant 1 & 2 1981626 3355787 Perfect match in all3 sub-barcodes 1645994 2790879 Perfect match in gene (40 bp) 10830132171930 % useful reads 45% 54% Total number of unique barcodecombination 16695 36595 Number of unique barcode combinations >30 80 281molecule Capture efficiency 0.03 0.04

Gene expression profiles for 100 unique barcode combinations with themost abundant molecules were determined for the high density and lowdensity samples. The gene expression profiles were generated based onthe sequencing results. FIG. 13A shows graphs of the gene expressionprofile for 35 of the 100 unique barcode combinations from the highdensity sample. For FIG. 13A, the number of unique molecules is on they-axis and the gene reference number is on the x-axis (see Table 6 forgenes corresponding to the gene reference number). FIG. 13A clearlydepicts general gene expression patterns for the mouse and Ramos cells.FIG. 13B-C show scatter plots of results based on principal componentanalysis of gene expression profile of the high density sample and lowdensity sample, respectively. Component 1, which is plotted on thex-axis, separates the two cell types. Component 2, which is plotted onthe y-axis, indicates variability in gene expression within the Ramoscell population. Each dot on the scatter plot represents one uniquebarcode combination, which is equivalent to one bead or one cell. Basedon the principal component analysis of the high density sample, 144beads corresponded to the mouse cells and 132 beads corresponded toRamos cells. Based on the principal component analysis of the lowdensity sample, 52 beads corresponded to the mouse cells and 27 beadscorresponded to Ramos cells.

Once the cell types were determined, cross-talk between the beads wasassessed by detecting the genes from Table 6 in the different celltypes. FIG. 13D-E depict graphs of the read per barcode (bc) combination(y-axis) versus the unique barcode combination, sorted by the totalnumber of molecules per bc combination (x-axis) for Ramos-like cells andmouse-like cells from the high density sample, respectively. FIG. 13F-Gdepict graphs of the number of molecules per bc combination (y-axis)versus the unique barcode combination, sorted by the total number ofmolecules per bc combination (x-axis) for Ramos-like cells andmouse-like cells from the high density sample, respectively. FIG. 13H-Idepict graphs of the read per barcode (bc) combination (y-axis) versusthe unique barcode combination, sorted by the total number of moleculesper bc combination (x-axis) for Ramos-like cells and mouse-like cellsfrom the low density sample, respectively. FIG. 13J-K depict graphs ofthe number of molecules per bc combination (y-axis) versus the uniquebarcode combination, sorted by the total number of molecules per bccombination (x-axis) for Ramos-like cells and mouse-like cells from thelow density sample, respectively. Table 8 shows the average foldcoverage or read redundancy per unique molecule for the low and highdensity samples.

TABLE 8 Low density High density Ramos-like Mouse-like Ramos-likeMouse-like Gene cells cells cells cells HS_CD74 29.75 3.17 23.75 2.15HS_CD79a 47.2 4.09 42.30 2.67 HS_IGJ 29.65 1.39 30.23 2.4 HS_TCL1A 45.742.26 39.00 4.13 HS_SEPT9 11.85 1.00 12.75 1.18 HS_CD27 37.99 1.00 32.121.10 HS_GAPDH 19.97 1.55 17.37 2.57 MM_B2M 1.21 31.98 3.05 31.48 MM_ACTM1.05 29.08 1.90 28.38 MM_HPRT 1.02 39.96 1.03 43.65 MM_SHDA 1.00 39.601.02 29.60

The results in Table 8 show that average fold coverage per uniquemolecule was much higher for human genes than mouse genes in Ramoscells, and vice versa.

As a control, a mixture of mouse and human cells were lysed in a tube,converted to cDNA synthesis with the beads, and the cDNA was sequenced.FIG. 4XL shows a graphical representation of the sequencing results. Asexpected, a large number of unique barcode (bc) combinations wasobserved, and most beads only had one to two copies total.

These results demonstrate that there was minimal cross-talk betweenbeads and that the cross-talk may be identified bioinformatically.

Example 8 Single Cell Nucleic Acid Library Production

The oligonucleotide conjugated supports disclosed herein may be used toproduce single cell nucleic acid libraries. In this example, single cellnucleic acid libraries are produced by adding a cell sample to a surface(e.g., grid) that has the oligonucleotide conjugated supports. Anoligonucleotide conjugated support comprises a plurality ofoligonucleotides conjugated to a bead. An oligonucleotide comprises (a)a cell label region comprising at least two distinct regions connectedby a linker; and (b) a molecular label region. Two or moreoligonucleotides on a bead comprise identical cell label regions. Two ormore oligonucleotides on a bead comprise two or more different molecularlabel regions. Two or more oligonucleotides on two or more differentbeads comprise two or more different cell label regions. Thus, each cellassociated with an oligonucleotide conjugated support has a differentcell label region. The concentration of cells in the cell sample issufficiently dilute to enable association of one or fewer cells to oneoligonucleotide conjugated support on the surface. Cells are lysed usinga lysis buffer. mRNAs from a cell are hybridized to the oligonucleotidesof the oligonucleotide conjugated support. Thus, all mRNAs from a cellare labeled with oligonucleotides comprising identical cell labelregions. Two or more mRNAs from a cell are labeled with two or moreoligonucleotides comprising two or more different molecular labelregions. A magnet is applied to the surface to purify theoligonucleotide conjugated solid supports from the surface. Theoligonucleotide conjugated solid supports may be individually purifiedfrom the surface. The mRNAs hybridized to the oligonucleotides on theoligonucleotide conjugated solid support are reverse transcribed toproduce labeled cDNA. The labeled cDNA comprise a reverse complement ofthe mRNA and a copy of the oligonucleotide that the mRNA was hybridizedto. The labeled cDNA are amplified by PCR to produce labeled amplicons.The labeled cDNA and/or labeled amplicons may be removed from the beadby restriction enzyme digestion. A library of nucleic acids from thesingle cell is produced from the labeled amplicons.

Alternatively, the oligonucleotide conjugated solid supports arepurified together. Reverse transcription of the mRNA may be performed onthe combined oligonucleotide conjugated solid supports. Because mRNAsfrom different cells are labeled with oligonucleotides comprisingdifferent cell label regions, the cell label regions may be used todetermine which cell the labeled cDNA or labeled amplicons originatedfrom. Thus, a library of nucleic acids from a plurality cells may beproduced, wherein the identity of the cell from which the labeledamplicon originated from may be determined by the cell label region.

Single cell nucleic acid libraries may also be produced by contactingthe cells with an agent prior to lysing the cell. The agent may be anantigen, drug, cell, toxin, etc. Thus, specialized single cell nucleiclibraries may be produced. Analysis of the nucleic acid libraries may beused to generate single cell drug expression profiles. Signaltransduction pathways on a single cell level may also be determined fromthese nucleic acid libraries. The nucleic acid libraries may also beused to determine the effects of antigens on specific cell types.

Example 9 Single Cell Expression Profiling

The oligonucleotide conjugated supports disclosed herein may be used todetermine the expression profile of single cells. In this example, acell sample comprising a mixture of cells is contacted with a pluralityof antibodies. A subset of the cells is purified using flow cytometry.The subset of cells is added to a microwell array. A plurality ofoligonucleotide conjugated supports is added to the microwell array. Anoligonucleotide conjugated support comprises a plurality ofoligonucleotides coupled to a nanoparticle. An oligonucleotide comprises(a) a cell label region comprising three distinct sequences connected bytwo predetermined sequences; and (b) a molecular label region. Two ormore oligonucleotides on a nanoparticle comprise identical cell labelregions. Two or more oligonucleotides on a nanoparticle comprise two ormore different molecular label regions. Two or more oligonucleotides ontwo or more different nanoparticles comprise two or more different celllabel regions. Thus, each cell associated with an oligonucleotideconjugated support has a different cell label region.

A magnet is applied to the microwell array and the cells that are notassociated with an oligonucleotide conjugated support are washed away. Asponge comprising a lysis buffer is placed on top of the microwellarray, thereby lysing the cells.

mRNAs from the lysed cells hybridize to the oligonucleotides on thebead. The mRNAs are reverse transcribed to produce labeled cDNA. Thelabeled cDNA comprise a reverse complement of the mRNA and a copy of theoligonucleotide that the mRNA was hybridized to. The labeled cDNA areamplified by PCR to produce labeled amplicons. The labeled amplicons aresequenced. Because each mRNA from a cell is labeled with the same celllabel and mRNAs from different cells are labeled with different celllabels, the sequence information of the labeled amplicons is used togenerate single cell expression profiles.

Example 10 Immunophenotyping by Single Cell Sequencing

A blood sample was collected from a subject and peripheral bloodmononuclear cells (PMBCs) were isolated from the blood sample. PMBCswere cultured in RPMI1640 medium and placed in an incubator overnight.The PMBCs were washed multiple times in PBS to remove the serum.Approximately 7000 PMBCs were deposited onto a microwell array with32,400 wells. Thus, most wells on the microwell array contained no cellsand some wells on the cell contained only 1 cell.Oligonucleotide-conjugated beads were applied to the microwell array.Each oligonucleotide-conjugated bead contained approximately 1 billionoligonucleotides attached to a bead. Each oligonucleotide attached tothe bead contained a 5′ amine, universal sequence, three-part cellularlabel (e.g., three cell label sections connected by two linkers),molecular label, and oligodT. Each bead contained a unique three-partcellular label, which is a result of the unique combination of the threecell label sections. All of the oligonucleotides on a single beadcontained the same three-part cellular label. Oligonucleotides fromdifferent beads contained different three-part cellular labels. Eachwell contained 1 or fewer oligonucleotide-conjugated bead. A cell lysisreagent was applied to the microwell array, resulting in lysis of thecells. Polyadenylated molecules (e.g., mRNA) from the cell hybridized tothe oligodT sequence of the oligonucleotides from theoligonucleotide-conjugated beads. The polyadenylated molecules that werehybridized to the oligonucleotides from the oligonucleotide-conjugatedbead were reverse transcribed with SuperScript II at 42° C. at 90minutes on a rotor. The oligonucleotide from theoligonucleotide-conjugated bead served as a primer for first strand cDNAsynthesis. A SMART oligo was incorporated in the cDNA synthesis suchthat the superscript II may add the complement of the SMART oligosequence to the 3′ end of the cDNA when it reaches the end. The cDNAsynthesis reaction produces a bead conjugated to unextendedoligonucleotides (e.g., oligonucleotides that were not attached to thepolyadenylated molecule from the cell) and the extended oligonucleotides(e.g., oligonucleotides that were attached to the polyadenylatedmolecule and comprise a polyadenylated molecule/cDNA hybrid).

The beads are combined and the oligonucleotides comprising thepolyadenylated molecule/cDNA hybrid were amplified. Multiplex PCR wasperformed to amplify a panel of 98 genes (see Table 9) from the cDNA onthe beads. Primers for the multiplexed PCR comprised a first genespecific primer that was designed to sit approximately 500 base pairsfrom the 3′ end of the mRNA and a nested gene-specific primer that wasdesigned to sit approximately 300 base pairs from the 3′ end of themRNA. Primers for the multiplex PCR were designed to require nosignificant complementarity in the last 6 bases of the primers in thepanel. If complementarity was detected in the multiplex PCR primers,then the primers were manually replaced. The multiplex PCR reactioncomprised the following steps: 1) 15 cycles of first gene specific PCR(KAPA multiplex mix, 50 nM of each primer—first gene specific primer anduniversal primer that is complementary to the universal sequence of theoligonucleotide-conjugated bead), Ampure clean up (0.7×bead to templateratio), 15 cycles of nested gene specific PCR (KAPA multiplex mix, 50 nMof each primer-nested gene-specific primer and universal primer that iscomplementary to the universal sequence of theoligonucleotide-conjugated bead), Ampure clean up (0.7×bead to templateratio), 8 cycles of final PCR to add full length Illumina adaptor (KAPAHiFi ReadyMix), and Ampure clean up (lx bead to template ratio).

TABLE 9 Gene Panel Cell type Gene Cell type Gene Cell type Gene Celltype Gene B cell PAX5 monocytes CD14 naïve CD62L (SELL) Th17 IL17A CD19classical S100A12 CD45RA IL17F CD20 monocytes CCR2 Naïve Th THPOK/ZBTB7BIL21 BCMA/ SELL/CD62L Naïve Tc RUNX3 IL22 TNFRSF17 (L-selectin) BAFFnonclassical CD16/FCGR3B memory CD45RO/PTPRC CCL20 TCL1A monocytesCX3CR1 CD44 IL23R TACI ITGAL Central CCR7 RORa/RORA B naïve IGHDconventional CD1b Memory TXK RORgamat/RORC IGHM myeoloid DC FOXQ1CD8+/CD4+ MBD2 Follicular T OX40L/TNFSF4/ helper CD252 B memory CD27CD209/ BCL6 CXCR5 DC-SIGN CD38 CD1e Effector BLIMP1 SLAM/SLAMF1 MemoryCD8+/CD4+ CD24 CCL17 Th1 CXCR3 ICOS AICDA DTNA IFNGR1 SAP/SH2D1A CD95plasmacytoid CLEC4C/ IL12RB2 Activated T CD69 dendritic cell CD303(rare) B transitional CD10 rare myeloid CD141/TM IFN gamma Activated TCD30 dendritic cell and B (0.02%) B reg IL10 NKT Th2 IL33R/IL1RL1Toll-like TLR1 plasma RASD1 PLZF/ZBTB16 IL4R receptors TLR2 AMPD1 SLAMF1CCR4 (innate) TLR3 SDC1 T cell CD3 (CD3D) CRTH2/PTGDR2 TLR4 (CD138) NKOSBPL5 CD3 (CD3E) IL4 TLR5 CD56/NCAM1 Cytotoxic T CD8 (CD8A) IL5 TLR6IGFBP7 CD8 (CD8B) Treg CD25 TLR7 KIR2DS5 PRF1 (perforin) FOXP1 TLR8KIR2DS2 EOMES TGFbeta TLR9 RAB4B Helper T CD4 IL10 TLR10

The amplified products were sequenced. The sequence reads with 150 bpwere aligned to entire mRNA sequences of the 98 genes listed in Table 9using Bowtie2. The results of the sequence alignment (see Table 10)demonstrate that the multiplex PCR reaction resulted in highly specificproducts. FIG. 14 shows a graph depicting the genes on the X-axis andthe log 10 of the number of reads. 16 genes of the 98 genes were notpresent. Absence of these genes may be due to the fact that some of thegenes target rare cells that may not be present in this blood sample.Overall, approximately, 84% of the genes from the 98 gene panel weredetected.

TABLE 10 total 6357075 aligned 0 times 703616 aligned exactly 1 time5584201 aligned >1 time 69258 % aligned exactly once 88%

Table 11 shows the results of the overall sequencing statistics. ForRead1, the total read1 match criteria required a perfect match to thethree-part cellular label (e.g., cell barcode) and at most 1 mismatch tothe linkers.

TABLE 11 total num read 6357075 total read1 match criteria 4384245 read2also align 3943667 % read2 align 89.95% number of unique cell bc 31129read count per unique bc >100 3228 read count per unique bc >50 3721 %useful reads 62.04%

FIG. 15A shows a graph of the distribution of genes detected perthree-part cell label (e.g., cell barcode). FIG. 15B shows a graph ofthe distribution of unique molecules detected per bead (expressing thegene panel).

Cell clustering analysis was performed to determine whether thesequencing results could be used to analyze cell populations based onthe cell barcode. SPADE (a minimum spanning tree algorithm developed bythe Nolan lab for CyTOF data) was used to cluster cells based on thepresence/absence of 17 genes. For a gene to be considered present, theaverage sequencing redundancy for the gen has to be greater than 5 fold.After sequence filtering, there were approximately 500 unique cellbarcodes (e.g., cell labels) associated with greater than 20 uniquemolecules. Each unique cell barcode corresponds to a single cell. Basedon the genes that were associated with a unique cell barcode, cells wereclustered into cell types. Table 12 shows a list of genes that may beused to definitively identify a cell type. Thus, cell barcodes that areassociated with CD20, IGHM, TCL1A and CD24 were designated as B-cells,whereas cell barcodes that are associated with CD8A, CD3D, CD3E, CD4 andCD62L were designated as T-cells. The remaining genes from Table 9 weremapped to the cell clusters. FIG. 16 depicts the cell clusters based onthe genes associated with a cell barcode. The size of the cluster isproportionate to the number of cells that were assigned to the cluster.The results shown in FIG. 16 demonstrate that the combination of celland molecular barcoding may be used to uniquely label copies ofmolecules from a single cell, which may enable immunophenotyping bysingle cell sequencing. In addition to clustering PMBCs into the majorcell types based on the genes listed in Table 12, the 98 gene panel mayalso be used to identify clusters of sub-types of the major cell types.Table 13 shows the frequency of each major cell type detected by singlecell sequencing. As shown in Table 13, with the exception of CD8+ Tcells, the percentage of each cell type corresponded to the normal cellpercentage range. A slightly higher percentage of CD8+ T cells wasobserved in the PMBC sample. Using the cell clusters based on FIG. 16,expression profiles of additional genes from the 98 gene panel were usedto further analyze the cell clusters.

TABLE 12 Major cell types Genes B cells CD20, IGHM, TCL1A, CD24 T cellsCD8A, CD3D, CD3E, CD4, CD62L NKT cells ZBTB16 Dendritic cells CD209Natural Killer cells KIR2DS5, KIR2DS2, CD16 Monocytes CD16, CD14, CCR2,S100A12, CD62L

TABLE 13 Cell type # cells percentage normal range monocytes 67 13.3%10-30% NK 85 16.9% up to 15% B 47 9.3% up to 15% CD8 210 41.7%  5-30%CD4 94 18.7% 25-60% total assigned cluster 503 100.0%

FIG. 17A-D show the analysis of monocyte specific markers. FIG. 17Eshows the cell cluster depicted in FIG. 16. FIG. 17A shows the cellexpression profile for CD14, which is a monocyte specific marker. “Hotcolors” (e.g., red) represent high gene expression and “cool colors”(e.g., blue) represent low gene expression. As shown in FIG. 17A, CD14is highly expressed in the monocyte population and had low to noexpression in the other cell types. The cell expression profile for CD16which is known to be present in both monocytes and NK is shown in FIG.17B. As shown in FIG. 17B, the monocyte and NK cell clusters had highexpression of CD16, whereas the other cell types had low to noexpression. CCR2 and S100A12 are known to be highly expressed inmonocytes. The CCR2 and S100A12 monocyte-specific expression was alsodemonstrated in the cell expression profiles shown in FIGS. 17C and D,respectively. However, the expression of CCR2 and S100A12 separated intotwo branches of monocyte cells. The other cell types exhibited low to noexpression of CCR2 and S100A12.

FIG. 18A-B show the analysis of the T cell specific markers. FIG. 18Cshows the cell cluster depicted in FIG. 16. FIG. 18A shows the cellexpression profile for CD3D which is a chain of the CD3 molecule. CD3 isa pan T cell marker. FIG. 18A shows that CD3D is highly expressed in twobranches of CD8+ T cells and moderately expressed in a third branch ofCD8+ T cells. However, CD3D is not highly expressed in CD4+ T cells.Also, the other cell types have low to no expression of CD3D. FIG. 18Bshows the cell expression profile for CD3E which is a chain of the CD3molecule. FIG. 18B shows that CD3D is highly expressed in CD4+ T cells.Different branches of CD8+ T cells exhibit high to moderate expressionof CD3D. Little to no expression of CD3D is observed in the other celltypes.

FIG. 19A-B show the analysis of the CD8+ T cell specific markers. FIG.19C shows the cell cluster depicted in FIG. 16. FIG. 19A shows the cellexpression profile for CD8A which is a chain of the CD8 molecule. Asshown in FIG. 19A, different branches of CD8+ T cells have variouslevels of CD8A expression, with some branches having high expression,other branches having moderate expression and one branch exhibiting lowto no expression of CD8A. High CD8A expression was observed in a branchof the CD16+NK cells. It has been reported in the literature that up to80% of NK cells express CD8. Little to no CD8A expression was observedin the other cell types. FIG. 19B shows the cell expression profile forCD8B which is a chain of the CD8 model. As shown in FIG. 19B, differentbranches of CD8+ T cells have various levels of CD8B expression, withone branch having high expression, some branches having moderateexpression and two branches exhibiting low to no expression of CD8B.High CD8B expression was also observed in a branch of the CD16+NK cells.Little to no CD8B expression was observed in the other cell types.

FIG. 20A shows the analysis of CD4+ T cell specific markers. FIG. 20Bshows the cell cluster depicted in FIG. 16. FIG. 20A shows theexpression profile for CD4. Moderate expression of CD4 was observed in asubset of cells in the CD4+ T cell cluster and high expression of CD4was observed in a branch of the monocyte cluster. It has previously beendocumented in the literature that monocytes also express CD4. Moderateto low expression of CD4 was observed in a branch of CD8+ T-cells and inNK cells. Low to no expression of CD4 was observed in the other celltypes.

FIG. 21A-D show the analysis of Natural Killer (NK) cell specificmarkers. FIG. 20E shows the cell cluster depicted in FIG. 16. FIG. 20Ashows the expression profile for KIR2DS2. All of the cell typesexhibited little to no KIR2DS2 expression. FIG. 20B shows the expressionprofile for KIR2DS5. Killer immunoglobulin receptors (KIRs) are known tobe expressed in NK cells and a subset of T cells. High expression ofKIR2DS5 was observed in 2 branches of NK cells and moderate to lowexpression of KIR2DS5 was observed in one branch of NK cells. Moderateto high expression of KIR2DS5 was observed in 2 branches of CD8+ Tcells. Low to no expression of KIR2DS5 was observed in all other celltypes. OSBPL5 and IGFBP7 are known to be highly expressed in NK cells.FIG. 20C shows the expression profile for OSBPL5. OSBPL5 was highlyexpressed in one branch of NK cells. Moderate to low expression ofOSBPL5 was observed in a branch of B cells. Low to no expression ofOSBPL5 was observed in all other cell types. FIG. 20D shows theexpression profile for IGFPBP7. High expression of IGFPBP7 was observedin two branches of NK cells and one branch of monocytes. Moderateexpression of IGFPBP7 was observed in one branch of B cells. Low to noexpression of IGFPBP7 was observed in all other cell types.

FIG. 22A-E show the analysis of B cell specific markers. FIG. 22F showsthe cell cluster depicted in FIG. 16. FIG. 22A shows the expressionprofile for IGHM CH4. IGHM CH4 was highly expressed in one branch of Bcells and moderately expressed in the second branch of B cells. Low tono expression of IGHM CH4 was observed in all other cell types. FIG. 22Bshows the expression profile for PAX5. PAX5 was highly expressed in onebranch of B cells. Low to no expression of PAX5 was observed in allother cell types. FIG. 22C shows the expression profile for CD20. CD20was highly expressed in one branch of B cells. Low to no expression ofCD20 was observed in all other cell types. FIG. 22D shows the expressionprofile for TCL1A. Low to no expression of TCL1A was observed in allother cell types. FIG. 22E shows the expression profile for IGHD CH2.IGHD CH2 was highly expressed in one branch of B cells. Low to noexpression of IGHD CH2 was observed in all other cell types.

FIG. 23A-F show the analysis of Toll-like receptors. Toll-like receptorsare mainly expressed by monocytes and some B cells. FIG. 23G shows thecell cluster depicted in FIG. 16. FIG. 23A shows the expression profilefor TLR1. One branch of monocytes exhibited high expression of TLR1 andtwo branches of monocytes exhibited moderate expression of TLR1. Low tono expression of TLR1 was observed in all other cell types. FIG. 23Bshows the expression profile for TLR4. One branch of monocytes exhibitedhigh expression of TLR4. Moderate TLR4 expression was observed in twobranches of monocytes and one branch of NK cells. Low to no expressionof TLR4 was observed in all other cell types. FIG. 23C shows theexpression profile for TLR7. High expression of TLR7 was observed in onebranch of monocytes and moderate expression of TLR7 was observed in onebranch of NK cells. Low to no expression of TLR7 was observed in allother cell types. FIG. 23D shows the expression profile for TLR2. Highexpression of TLR2 was observed in one branch of B cells. Low to noexpression of TLR2 was observed in all other cell types. FIG. 23E showsthe expression profile for TLR3. High expression of TLR3 was observed inone branch of B cells. Low to no expression of TLR3 was observed in allother cell types. FIG. 23F shows the expression profile for TLR8. Highexpression of TLR8 was observed in three branches of monocytes. Moderateto low expression of TLR8 was observed in two branches of monocytes andone branch of NK cells. Low to no expression of TLR8 was observed in allother cell types.

These results demonstrate that massively parallel single cell sequencingmay successfully identify major cell types in PMBCs. The sequencingresults also determined that some cell markers that are used in FACs foridentifying cell types do not have high mRNA expression (e.g., CD56 forNK cells, CD19 for B cells). In addition, many of the genes in the genepanel were expressed across multiple cell types. These expressionprofiles may be used to subtype cells within a major cell type (e.g.,activated cell versus resting cell, etc.).

Example 11 Identifying Rare Cells in a Population

In this experiment, massively parallel single cell sequencing is used toidentify cancer cells from a mixture of cancer and non-cancer cells.Ramos (Burkitt lymphoma) cells were spiked into a population of CD19+ Bcells that were isolated from a healthy individual. The concentration ofthe Ramos cells in the mixed population was about 4-5%. Approximately7000 normal B cells and 300 Ramos cells were deposited on a microwellarray with 25,200 wells. Thus, most wells on the microwell arraycontained no cells and some wells on the cell contained only 1 cell.Oligonucleotide-conjugated beads were applied to the microwell array.Each oligonucleotide-conjugated bead contained approximately 1 billionoligonucleotides attached to a bead. Each oligonucleotide attached tothe bead contained a 5′ amine, universal sequence, three-part cellularlabel (e.g., three cell label sections connected by two linkers),molecular label, and oligodT. Each bead contained a unique three-partcellular label, which is a result of the unique combination of the threecell label sections. All of the oligonucleotides on a single beadcontained the same three-part cellular label. Oligonucleotides fromdifferent beads contained different three-part cellular labels. Eachwell contained 1 or fewer oligonucleotide-conjugated bead. A cell lysisreagent was applied to the microwell array, resulting in lysis of thecells. Polyadenylated molecules (e.g., mRNA) from the cell hybridized tothe oligodT sequence of the oligonucleotides from theoligonucleotide-conjugated beads. The polyadenylated molecules that werehybridized to the oligonucleotides from the oligonucleotide-conjugatedbead were reverse transcribed with SuperScript II at 42° C. at 90minutes on a rotor. The oligonucleotide from theoligonucleotide-conjugated bead served as a primer for first strand cDNAsynthesis. A SMART oligo was incorporated in the cDNA synthesis suchthat the superscript II may add the complement of the SMART oligosequence to the 3′ end of the cDNA when it reaches the end. The cDNAsynthesis reaction produces a bead conjugated to unextendedoligonucleotides (e.g., oligonucleotides that were not attached to thepolyadenylated molecule from the cell) and the extended oligonucleotides(e.g., oligonucleotides that were attached to the polyadenylatedmolecule and comprise a polyadenylated molecule/cDNA hybrid).

The beads are combined and the oligonucleotides comprising thepolyadenylated molecule/cDNA hybrid were amplified. Multiplex PCR wasperformed to amplify a panel of 111 genes from the cDNA on the beads.The 111 genes represent markers for different subsets of B cells.Primers for the multiplexed PCR comprised a first gene specific primerthat was designed to sit approximately 500 base pairs from the 3′ end ofthe mRNA and a nested gene-specific primer that was designed to sitapproximately 300 base pairs from the 3′ end of the mRNA. Primers forthe multiplex PCR were designed to require no significantcomplementarity in the last 6 bases of the primers in the panel. Ifcomplementarity was detected in the multiplex PCR primers, then theprimers were manually replaced. The multiplex PCR reaction comprised thefollowing steps: 1) 15 cycles of first gene specific PCR (KAPA multiplexmix, 50 nM of each primer-first gene specific primer and universalprimer that is complementary to the universal sequence of theoligonucleotide-conjugated bead), Ampure clean up (0.7×bead to templateratio), 15 cycles of nested gene specific PCR (KAPA multiplex mix, 50 nMof each primer-nested gene-specific primer and universal primer that iscomplementary to the universal sequence of theoligonucleotide-conjugated bead), Ampure clean up (0.7×bead to templateratio), 8 cycles of final PCR to add full length Illumina adaptor (KAPAHiFi ReadyMix), and Ampure clean up (1×bead to template ratio).

The amplified products were sequenced. The sequence reads comprising 150bp were aligned to entire mRNA sequences of the 111 genes (Table 17)using Bowtie2. The results of the sequence alignment (see Table 14)demonstrate that the multiplex PCR reaction resulted in highly specificproducts. FIG. 24 depicts a graph of the genes versus the log 10 of thenumber of reads. 24 of the 111 genes were not present. At least two ofthe genes, RAG1 and RAG2 which are involved in VDJ recombination andshould be present only in pre-B cells, should not be present. A few ofthe absent genes are specific for plasma cells, which are very rarelypreserved in frozen cells.

TABLE 17 CD19 AURKB FOXP1 CCND3 TLR1 FOXP3 CXCL12 GNAI2 CD27 CD81 MCL1IL12A TLR2 LAG3 CCL3 RGS1 CD138 CD80 IFNB1 IFNG TLR3 CD73 CCL14 CD5 CD38CD23a BLNK TNFA TLR4 CD70 CCL20 CD22 CD24 CD44 CD40LG IL2 TLR5 CCR7CCL18 PIK3CD CD10 LEF1 IGBP1 IL4 TLR6 CD45RA TCL1A DOCK8 CD95 CXCR5 IRF4IL6 TLR7 PDCD1 TACI CD11b CD21 PRKCB CD79a BAFF TLR8 MYC AICDA FCGR2BCXCR3 PRKCD LTA IGHE TLR9 CD25 FCRL4 CD72 CD40 CD20 HDAC5 IGHD TLR10FCAMR BCL2 BCL11B CD69 CD30 RAG1 IGHM GAPDH CCND2 FASLG CD86 CD1c CD30LRAG2 IGHA CD9 MKI67 BCL6 TBX21 IL10 BAFFR CD1d IGHG1 CD11c IL21R IGHG2PRDM1 IL4R CMRF- TGFB1 IGHG4 IL6R HLA- IGHG3 35H DRA

TABLE 14 total 5711013 aligned 0 times 504775 aligned exactly 1 time5203308 aligned >1 time 2930 % aligned exactly once 91.6%

Table 15 shows the results of the overall sequencing statistics. ForRead1, the total read1 match criteria required a perfect match to thethree-part cellular label (e.g., cell barcode) and at most 1 mismatch tothe linkers.

TABLE 15 total num read 5711013 total read1 match criteria 3795915 read2also align 3495392 % read2 align 92% number of unique cell bc 40764 readcount per unique bc >100 3313 read count per unique bc >50 4154 % usefulreads 61%

FIG. 25A-D shows graphs of the molecular barcode versus the number ofreads or log 10 of the number of reads for two genes. FIG. 25A shows agraph of the molecular barcode (sorted by abundance) versus the numberof reads for CD79. FIG. 25B shows a graph of the molecular barcode(sorted by abundance) versus the log 10 of the number of reads for CD79.FIG. 25C shows a graph of the molecular barcode (sorted by abundance)versus the number of reads for GAPDH. FIG. 25D shows a graph of themolecular barcode (sorted by abundance) versus the log 10 of the numberof reads for GAPDH.

856 cells were retained for analysis. FIG. 26A shows a graph of thenumber of genes in the panel expressed per cell barcode versus thenumber of unique cell barcodes/single cell. FIG. 26B shows a histogramof the number of unique molecules detected per bead versus frequency ofthe number of cells per unique cell barcode carrying a given number ofmolecules. A small subset of cells showed distinctly higher number ofmRNA molecules and number of genes expressed from the 111 gene panel(see circled sections in FIG. 26A-B). FIG. 26C shows a histogram of thenumber of unique GAPDH molecules detected per bead versus frequency ofthe number of cells/unique cell barcode carrying a given number ofmolecules.

Principal component analysis (PCA) was used to generate a scatterplot ofcells. FIG. 27 shows a scatterplot of the 856 cells. PCA identified thesmall subset of cells with a different gene expression pattern than themajority of cells. The subset of cells contained 18 cells, which isapproximately 2% of all of the cells analyzed. This percentage issimilar to the percentage of Ramos cells that was spiked into thepopulation.

Ramos cells are derived from follicular B cells and strongly express Bcell differentiation markers CD20, CD22, CD19, CD10 and BCL6. Ramoscells also express IgM and overexpress c-myc. FIG. 28 shows a heat mapof expression of the top 100 (in terms of the total number of moleculesdetected). The subset of cells (18 cells) that express much higherlevels of mRNA also strongly express genes that are known markers forRamos cells (e.g., CD10, Bcl-6, CD22, C-my, and IgM).

These results demonstrate that massively parallel single cell sequencingsuccessfully identified small subsets (as low as 2%) of abnormal celltypes in a cell suspension. Massively parallel single cell sequencingmay be used in cancer diagnostics (e.g., biopsy/circulating tumorcells). Since cancer cells are larger in size and carry more mRNA, theymay be easily differentiated from normal cells.

Example 12 Massively Parallel Single Cell Whole Genome and MultiplexAmplification of gDNA Targets Using RESOLVE

FIG. 29 shows a workflow for this example. As shown in FIG. 29, a cellsuspension is applied to a microwell array (2901). The number of cellsin the cell suspension is less than the number of wells in the microwellarray, such that application of the cell suspension to the microwellarray results in a well in the microwell array containing 1 or fewercells. Oligonucleotide-conjugated beads (2905) are applied to themicrowell array. An oligonucleotide-conjugated bead (2905) contains abead (2910) attached to an oligonucleotide comprising a 5′ amine (2915),universal primer sequence (2920), cell label (2925), molecular label(2930) and randomer (2935). The oligonucleotide-conjugated bead containsapproximately 1 billion oligonucleotides. An oligonucleotide contains a5′ amine, universal primer sequence, cell label, molecular label, andrandomer. Each oligonucleotide on a single bead contains the same celllabel. However, two or more oligonucleotides on a single bead maycontain two or more different molecular labels. A bead may containmultiple copies of oligonucleotides containing the same molecular label.

After the oligonucleotide-conjugated beads are added to the microwellarray, a cell lysis buffer is applied to the array surface. As shown inFIG. 29, the genomic DNA (2945) from the cell hybridizes to the randomersequence (2935) of the oligonucleotide-conjugated beads (2940). Aneutralization buffer is added to the array surface. A DNA polymerase(e.g., Phi29) and dNTPs are added to the array surface. The randomersequence (2935) acts as a primer for amplification of the genomic DNA,thereby produce a gDNA-conjugated bead (2555). The gDNA-conjugated bead(2955) contains an oligonucleotide comprising a 5′ amine (2915),universal primer sequence (2920), cell label (2925), molecular label(2925), randomer (2935) and copy of the genomic DNA (2955). The originalgenomic DNA (2945) is hybridized to the randomer (2935) and the copy ofthe genomic DNA (2955). For a single bead, there are multiple differentgenomic DNA molecules attached to the oligonucleotides.

As shown in FIG. 29, the gDNA-conjugated beads (2950) from the wells arecombined into an eppendorf tube (2960). The genomic DNA on the gDNA MDAmix containing randomers, dNTPs and a DNA polymerase (e.g., Phi29) isadded to the eppendorf tube containing the combined gDNA-conjugatedbeads. The labeled genomic DNA is further amplified to yield labeledamplicons (2965) in solution. A labeled amplicon (2965) comprises auniversal primer sequence (2920), cell label (2925), molecular label(2930), randomer (2935), and copy of the genomic DNA (2955). The labeledamplicons are sheared to smaller pieces of approximately lkb or less.Alternatively, the labeled amplicons may be fragmented by toTagmentation (Nextera). Shearing or fragmenting the labeled ampliconsresults in labeled-fragments (2980) and unlabeled fragments (2985). Thelabeled fragment (2980) contains the universal primer sequence (2920),cell label (2925), molecular label (2930), randomer (2935), and fragmentof the copy of the genomic DNA (2955). Adaptors (2970, 7975) are addedto the fragments. The universal primer sequence may be used to selectfor labeled fragments (2980) via hybridization pulldown or PCR using theuniversal primer sequence and a primer against one of the adaptors(2970, 2975).

The labeled fragments may be sequenced. Sequence reads comprising asequence of the cell label, molecular label and genomic fragment may beused to identify cell populations from the cell suspension. Principalcomponent analysis may be used to generate scatterplots of the cellsbased on known cell markers. Alternatively, or additionally, SPADE maybe used to produce cell cluster plots. A computer software program maybe used to generate a list comprising a cell label and the molecularlabels and genomic fragments associated with the cell label.

Example 13 Massively Parallel Sequencing to Identify Cells in aHeterogeneous Population

The experimental workflow for this example is shown in FIG. 30. As shownin FIG. 30, a mixed population of cells was stochastically dispersedonto a microwell array. In this example, the mixed population of cellscomprises a mixture of Ramos cells and K562 cells. The cell suspensioncomprises a low concentration of cells such that each microwell in thearray contains 1 or fewer cells. After the cells were applied to themicrowell array, a plurality of oligonucleotide conjugated beads wasstochastically dispersed onto the microwell array. The oligonucleotidebead contains a plurality of oligonucleotides comprising a 5′ amine,universal primer sequence, cell label, molecular label, and oligodT. Thecell labels of the plurality of oligonucleotides from a single bead areidentical. A single bead may comprise multiple oligonucleotidescomprising the same molecular label. In addition, a single bead maycomprise multiple oligonucleotides comprising different molecularlabels. A cell label of an oligonucleotide conjugated to a first bead isdifferent from a cell label of an oligonucleotide conjugated to a secondbead. Thus, the cell label may be used to differentiate two or moreoligonucleotide conjugated beads. The cells were lysed and the RNAmolecules from a single cell were attached to the oligonucleotideconjugated beads in the same well. FIG. 30 shows the attachment of thepolyA sequence of a RNA to the oligodT sequence of the oligonucleotide.After attachment of the RNA molecules from the individual cells to theoligonucleotide conjugated beads in the same well, the beads werecombined into a single sample. A cDNA synthesis reaction was carried outon the beads in the single sample. FIG. 30 shows the product of the cDNAsynthesis comprises a bead attached to an oligonucleotide, theoligonucleotide comprising the 5′ amine, universal primer sequence, celllabel, molecular label, oligodT and a copy of the RNA molecule. Forsimplicity, only one oligonucleotide is depicted in FIG. 30, however, inthis example, each oligonucleotide conjugated bead comprisesapproximately 1 billion oligonucleotides. As shown in FIG. 30,multiplexed PCR was performed with the beads in the single sample usinga universal primer that hybridized to the universal primer sequence anda gene-specific primer that hybridized to the copy of the RNA molecule.The gene-specific primers were designed to bind to Ramos-specific genesor K562-specific genes from the gene panel shown in Table 16. As acontrol, a GAPDH gene-specific primer was also used in the multiplexedPCR reaction. Lastly, next-generation sequencing was used to sequencethe amplified products. The sequencing reads included informationpertaining to the cell label, molecular label and the gene. Usingprincipal component analysis, a scatter plot of the cells wasconstructed based on the sequencing information pertaining to the celllabel, molecular label and the gene. Analogous to how FACs is used tosort cells and scatter plots based on the surface markers is used togroup cells, the cell label is used to identify genes from a single celland the molecular label is used to determine the quantity of the genes.This combined information is then used to relate the gene expressionprofile individual cells. As shown in FIG. 31A, massively parallelsingle cell sequencing with cell and molecular labels was able tosuccessfully identify the two cell populations (K562 and Ramos cells) inthe mixed cell population.

TABLE 16 Gene Cell Gene Cell CD74 Ramos specific CD41 K562 specificCD79a Ramos specific GYPA K562 specific IGJ Ramos specific GATA2 K562specific TCL1A Ramos specific GATA1 K562 specific SEPT9 Ramos specificHBG1 K562 specific CD27 Ramos specific GAPDH Common

Example 14 Massively Parallel Single Cell Sequencing with PrincipalComponent Analysis

In this example, mRNA molecules from individual cells werestochastically labeled with oligonucleotide conjugated beads inparallel. PBMCs were isolated from blood and frozen at 80° C. inRPMI1640 plus FBS and DMSO. The PMBCs were thawed and washed three timeswith PBS. A PBMC sample comprising a mixture of cell types (4000 totalcells) was stochastically applied to an agarose microwell array. Theagarose microwell array contained 37,500 cells. A mixture of 150,000oligonucleotide conjugated beads was stochastically applied to themicrowell array via a PDMS gasket that surrounded the microwell array.The oligonucleotide conjugated bead is depicted in FIG. 1. Forsimplicity, only one oligonucleotide is shown to be attached to thebead, however, the oligonucleotide conjugated beads containedapproximately 1 billion oligonucleotides.

Cells were lysed by placing the microwell array on a cold block for 10minutes and by applying lysis buffer to the array surface. Once thecells in the wells were lysed, the mRNA molecules from the single cellswere attached to the oligonucleotide conjugated bead via the oligodTsequence. A magnet was applied to the array and the array was washedtwice with wash buffer.

The beads with the attached mRNA molecules were combined into aneppendorf tube. The mRNA molecules attached to the beads were reversetranscribed to produce cDNA. The following cDNA synthesis mixture wasprepared as follows:

Component Volume (uL) Water 8 dNTP (10 mM) 2 5x first strand buffer 4MgCl2 2.4 SuperRase In 1 SMART oligo (50 uM) 0.4 0.1M DTT 1 100× BSA 0.2SSII 1 total 20

The cDNA synthesis mixture was added to the eppendorf tube containingthe beads with the attached mRNA molecules. The eppendorf tube wasincubated at 40° C. for 90 minutes on a rotor. The cDNA synthesisreaction occurred on the beads. After 90 minutes, a magnet was appliedto the tube and the cDNA mix was removed and replaced with the followingExoI reaction mixture:

Component Volume (uL) ExoI buffer 2 water 17 ExoI 1

The tubes were incubated at 37° C. for 30 minutes on a rotor. The tubeswere then transferred to a thermal cycler for 15 minutes at 80° C. Afterincubating the tube at 80° C. for 15 minutes, 70 microliters ofTE+Tween20 was added to the tube. A magnet was applied to the tube andthe buffer was removed. The beads were then resuspended in 50microliters TE+Tween20.

The cDNA attached to the beads were amplified by real-time PCR using thefollowing amplification mixture:

Component Volume (uL) 2× iTaq mix 10 GAPDH ILMN (10 uM) 0.6 ILR2 (10 uM)0.6 bead 2 water 6.8 total 20

The labeled cDNA amplicons were sequenced to detect the cell label,molecular index, and gene. Sequencing reads were aligned to the celllabel, then the gene, and lastly the molecular label. A cell labelassociated with 4 or more genes or associated with 10 or more uniquetranscript molecules, with each unique transcript molecule sequencedmore than once, was designated a cell. Principal component analysis withall of genes from Table 9 detected was used to identify the set of genesthat had the greatest contribution to the variation in data. 632 singlecells were used in the principal component analysis. Based on thesequencing results, 81 out of the 98 genes were detected.

FIG. 32 shows a principal component analysis plot for GAPDH expression.As shown in FIG. 32, two cell clusters were observed based on thelocation of the principal component space.

FIG. 33A-F shows the principal component analysis (PCA) for monocyteassociated genes. FIG. 33A shows the PCA for CD16. FIG. 33B shows thePCA for CCRvarA. FIG. 33C shows the PCA for CD14. FIG. 33D shows the PCAfor S100A12. FIG. 33E shows the PCA for CD209. FIG. 33F shows the PCAfor IFNGR1.

FIG. 34A-B shows the principal component analysis (PCA) for pan-T cellmarkers (CD3). FIG. 34A shows the PCA for CD3D and FIG. 34B shows thePCA for CD3E.

FIG. 35A-E shows the principal component analysis (PCA) for CD8 T cellassociated genes. FIG. 35A shows the PCA for CD8A. FIG. 35B shows thePCA for EOMES. FIG. 35C shows the PCA for CD8B. FIG. 35D shows the PCAfor PRF1. FIG. 35E shows the PCA for RUNX3.

FIG. 36A-C shows the principal component analysis (PCA) for CD4 T cellassociated genes. FIG. 36A shows the PCA for CD4. FIG. 36B shows the PCAfor CCR7. FIG. 36C shows the PCA for CD62L.

FIG. 37A-F shows the principal component analysis (PCA) for B cellassociated genes. FIG. 37A shows the PCA for CD20. FIG. 37B shows thePCA for IGHD. FIG. 37C shows the PCA for PAX5. FIG. 37D shows the PCAfor TCL1A. FIG. 37E shows the PCA for IGHM. FIG. 37F shows the PCA forCD24.

FIG. 38A-C shows the principal component analysis (PCA) for NaturalKiller cell associated genes. FIG. 38A shows the PCA for KIR2DS5. FIG.38B shows the PCA for CD16. FIG. 38C shows the PCA for CD62L.

Based on the principal component analyses, monocytes and lymphocytesformed two distinct clusters on PC1. B, T, and NK cells formed anothercluster that resided as a continuum in the cluster along PC2. FIG. 39shows the PCA analysis of GAPDH expression with annotations for the celltypes and cell subtypes. FIG. 40 depicts a heat map that shows thecorrelation in gene expression profile between cells. Along the diagonalstarting with the left upper corner, the cells are monocytes, naive CD4T cells, naive CD8 T cells, cytotoxic CD8 T cells, NK cells, and Bcells. FIG. 41 shows another version of a heat map demonstrating thecorrelation between gene expression and cell type. FIG. 42 shows a heatmap demonstrating the correlation in gene expression profile betweengenes.

Example 15 Uncovering Cellular Heterogeneity by Digital Gene ExpressionCytometry

An approach for gene expression cytometry is presented combiningnext-generation sequencing with stochastic barcoding of single cells.Thousands of cells were deposited randomly onto an array ofapproximately 150,000 microwells. A library of beads bearing cell- andtranscript-barcoding capture probes was added so that each cell ispartitioned alongside a bead with a unique cell barcode. Following celllysis, mRNAs were hybridized to beads, and were pooled for reversetranscription, amplification, and sequencing. The digital geneexpression profile for each cell was reconstructed when barcodedtranscripts were counted and assigned to the cell of origin. We appliedthe technology to dissect the human hematopoietic system into cellsub-populations, and to characterize the heterogeneous response ofimmune cells to in vitro stimulation. Furthermore, the high sensitivityof the method was demonstrated by the detection of rare cells, such asantigen-specific T cells, and tumor cells in a high background of normalcells.

Introduction

Understanding cellular diversity and function in a large collection ofcells requires the measurement of specific genes or proteins expressedby individual cells. Flow cytometry is well established for measuringprotein expression of single cells, yet mRNA expression measurements aretypically conducted in bulk samples, obscuring individual cellcontributions. While single cell mRNA expression measurements usingmicrotiter plates or commercial microfluidic chips have recently beenreported (1-5), these approaches are extremely low-throughput anddifficult to scale. Because of these limitations, most studies to dateare restricted in both the number of cells interrogated and the numberof conditions explored.

Here, we have developed a highly scalable approach that enables routine,digital gene expression profiling of thousands of single cells across anarbitrary number of genes. Microscale engineering and combinatorialchemistry were used to label all mRNA molecules in a cell with a uniquecellular barcode in a massively parallel manner. In addition, eachtranscript copy within a cell was tagged with a molecular barcode,allowing absolute digital gene expression measurements (6). Tagged mRNAmolecules from all cells were pooled, amplified, and sequenced. Thedigital gene expression profile of each cell was reconstructed using thecell and molecular barcodes on each sequence. This highly scalabletechnology enables gene expression cytometry, which we term CytoSeq. Wehave applied the technique to multiparameter genetic classification ofthe hematopoietic system and demonstrated its use for studying cellularheterogeneity and detecting rare cells in a population.

Results

CytoSeq

The procedure was outlined in FIG. 43A. A cell suspension was firstloaded onto a microfabricated surface with up to 150,000 microwells.Each 30 micron diameter microwell has a volume of .about.20 picoliters.The number of cells was adjusted so that only .about.1 out of 10 or morewells receives a cell. The cells settled within the wells by gravity.

Magnetic beads were loaded onto the microwell array to saturation, suchthat a bead sat partially on top of, or adjacent to, each cell within awell. The dimension of the bead was chosen such that each microwell mayhold only one bead. Each magnetic bead carried approximately one billionoligonucleotide templates with the structure outlined in FIG. 43B. Eacholigonucleotide displayed a universal priming site, followed by a celllabel, a molecular label, and a capture sequence of oligo(dT). All theoligonucleotides on each bead have the same cell label but contain adiversity of molecular labels. We have devised a combinatorialsplit-pool method to synthesize beads with a diversity of close to onemillion. The probability of having two single cells being tagged withthe same cell label was low (on the order of 10⁴) because only.about.10% of the wells were occupied by a single cell. Similarly, thediversity of the molecular labels on a single bead was on the order of10⁴, and the likelihood of two transcript molecules of the same gene inthe same cell being tagged with the same molecular label was also low.

Lysis buffer was applied onto the surface of the microwell array anddiffuses into the microwells. The poly(dA) tailed mRNA moleculesreleased from a cell hybridize to the oligo(dT) on the 3′ end of theoligonucleotides on the bead. Because the cell was adjacent to the bead,under the high salt conditions of the lysis buffer and high localconcentration of mRNA (tens of nanomolar), mRNA molecules were capturedon the bead.

After lysis and hybridization, all beads were collected from themicrowell array into a tube using a magnet. From this point forward, allreactions were carried out in a single tube. cDNA synthesis wasperformed on the beads using conventional protocols (Methods). The cDNAmolecules derived from each cell were covalently attached to theircorresponding bead, each tagged on the 5′ end with a cell label and amolecular label. Nested multiplex PCRs were carried out to amplify genesof interest (FIG. 55). Because the mRNA from each cell had been copiedonto a bead as cDNA, the beads may be repeatedly amplified and analyzed,for example, for a different set of genes.

Sequencing of the amplicons revealed the cell label, the molecularlabel, and the gene identity (FIG. 55). Computational analysis groupedthe reads based on the cell label, and collapsed the reads with the samemolecular label and gene sequence into a single entry to suppress anyamplification bias. The use of molecular label enabled us to measure theabsolute number of molecules per gene per cell, and therefore allowedthe direct comparison of cellular expression level across biologicalsamples that may have undergone different depths of sequencing.

Identification of Distinct Cell Types in Controlled Cell Mixtures

In order to measure the ability of the method to separate two celltypes, a .about.1:1 mixture of K562 and Ramos cells was loaded onto themicrowell array with 10,000 wells. Approximately 6000 cells were used tocapture 1000 cells. A panel of 12 genes was selected and amplified fromthe beads. The panel consists of 5 genes specific for K562 (myelogenousleukemia) cells, 6 genes specific for Ramos (follicular lymphoma) cells,and the housekeeping gene GAPDH (Table 18). With approximately 1000cells captured on a 10,000-well array each with a single bead, only 10%of the beads should carry mRNA and one should in theory observe only amaximum of 1000 unique cell labels in the sequencing data. Indeed, wefound 768 cell labels that were associated with significant number ofreads after data filtering (see Methods for filtering criteria). As acomparison, we carried out bulk cell lysis and mRNA capture in amicrocentrifuge tube with similar number of cells and beads, andobserved a large number of cell labels with mostly only one readassociated with each cell label. This demonstrates that the microwellarray was effective in confining hybridization of mRNA from a singlecell to the bead in the same well.

The gene expression profile of each of the 768 single cells wasclustered using principal component analysis (PCA) (FIG. 31A). The firstprincipal component (PC) clearly separated the single cells into twomajor clusters based on the cell type. The genes that contributed to thepositive side of the first principal component were those that arespecific to Ramos, while the genes that contributed to the negative sideof the same principal component were those that are specific to K562.This successful clustering of cells into groups based on their specificexpression showed that inter-well contamination, if any, was negligible.The second principal component highlighted the high degree ofvariability in fetal hemoglobin (HBG1) within the K562 cells, which hadbeen observed previously (7).

TABLE 18 Nested Primer  SEQ ID with Common SEQ ID Gene Outer Primer NO:5′ Flanking Sequence NO: CD41 CCCCTGGAAGAA  2 CAGACGTGTGCTCTTCCGATCTT  3GATGATGA TCTCCAACAAGTTGCCTCC GYPD GAGGAAATGAAG  4CAGACGTGTGCTCTTCCGATCTA  5 CCAAACACA ATCGTGACCTTAAAGGCCC GATA1TTAGCCACCTCA  6 CAGACGTGTGCTCTTCCGATCTC  7 TGCCTTTC TACTGTGGTGGCTCCGCTGATA2 GGAGGAGGATTG  8 CAGACGTGTGCTCTTCCGATCTG  9 TGCTGATGTGTCCGCATAAGAAAAAGAATC HBG1 GCAAGAAGGTGC 10 CAGACGTGTGCTCTTCCGATCTC 11TGACTTCC TGCATGTGGATCCTGAGAA CD27 CTGCAGTCCCAT 12CAGACGTGTGCTCTTCCGATCTG 13 CCTCTTGT ATGAGGTGGAGAGTGGGAA IGJ GGACATAACAGA14 CAGACGTGTGCTCTTCCGATCTC 15 CTTGGAAGCA AATCCATTTTGTAACTGAACCTT TCL1AAAGCCTCTGGGT 16 CAGACGTGTGCTCTTCCGATCTT 17 CAGTGGT GGAAAAGGGATAGAGGTTGGCD74 TAGACAGATCCC 18 CAGACGTGTGCTCTTCCGATCTA 19 CGTTCCTGCAGGGAGAAGGGATAACCC SEPT9 CAGCATCCCAGC 20 CAGACGTGTGCTCTTCCGATCTC 21CTTGAG CTCAATGGCCTTTTGCTAC CD79a CCTCTAAACTGC 22 CAGACGTGTGCTCTTCCGATCTC23 CCCACCTC CTTAATCGCTGCCTCTAGG GAPDH CACATGGCCUCC 24CAGACGTGTGCTCTTCCGATCTC 25 AAGGAGUAA AGCAAGAGCACAAGAGGAA

In another experiment, we spiked in Ramos (Burkitt lymphoma) cells at afew percentage into primary B cells from a healthy individual. A panelof 111 genes (Table 22) was designed to represent different states of Bcells. 1198 single cells were analyzed. A small group of the population,constituting 18 single cells (.about.1.5% of the population), was foundto have a distinct gene expression pattern as compared to the rest (FIG.31B). The genes that were preferentially expressed by this group areknown to be associated with Burkitt lymphoma, such as MYC and IgM, aswell as B cell differentiation markers (CD10, CD20, CD22, BCL6) that areexpressed specifically by follicular B cells, which are the subset of Bcells that Burkitt lymphoma originates (FIGS. 31C and 31D). In addition,this group of cells carried higher level of CCND3 and GAPDH, as well asan overall higher mRNA content, as determined by the total number ofunique mRNA molecules detected based on analyzing the molecular indices(FIG. 31B). This finding was consistent with the fact that lymphomacells are physically larger than the primary B cells in normalindividuals, and that they are rapidly proliferating and producinglarger amount of transcripts.

Simultaneous Identification of Multiple Cell Types in Human PBMCs

While the controlled experiments involved artificial mixtures of twodistinct types of cells, most naturally occurring biological samplescontain diverse populations with numerous cell types and states withmore subtle differences in gene expression profile. A prominent exampleis blood. We carried out an experiment in which we aimed tosimultaneously identify all of the major cell types in human peripheralblood mononuclear cells (PBMCs), including monocytes, NK cells, and thedifferent T and B cell subsets, by measuring the expression profile of apanel of 98 genes (Table 19) that are specific to each of the major celltype. Unlike traditional immunophenotyping that is limited mostly tosurface protein markers, we included genes that encode cytokines,transcription factors, and intracellular proteins of various cellularfunctions in addition to surface proteins. We analyzed with PCA thedigital gene expression profile of 632 single PBMCs using 81 genespresent (FIG. 32-39). The first principal component clearly separatedmonocytes and lymphocytes into two orthogonal clusters, as evidenced bythe expression of CD16a, CD14, S100A12, and CCR2 in one cluster, andlymphocyte associated genes in the other. The different subtypes oflymphocytes lay in a continuum along the second principal component,with B cells (expressing IgM, IgD, TCL1A, CD20, CD24, PAX5) at one end,naive T cells (expressing CD4, CCR7, CD62L) in the middle, and cytotoxicT cells (expressing CD8A, CD8B, EOMES, PRF1) at the other end. Naturalkiller cells that express killer-like immunoglobulin receptor, CD16a,and perforin (PRF1) lay in the space between monocytes and cytotoxic Tcells. We also observed that GAPDH, an indicator of cellular metabolism,was expressed at highest levels in monocytes and lowest in B cells,which are presumably mostly resting. Correlation analysis of geneexpression profile across cells reiterated observations with PCA andrevealed additional smaller subsets of cells within each major cell type(FIG. 40A-B). A replicate experiment of the same PBMC sample with 731cells yielded largely similar segregation and cell type frequency (FIG.41).

TABLE 22 SEQ ID Nested Primer with Common  SEQ ID Gene Outer Primer NO:5′ Flanking Sequence NO: CD19 GCAGGGTCCCAG 26 CAGACGTGTGCTCTTCCGATCTCC27 TCCTATG AATCATGAGGAAGATGCA CD27 TCCAGGAGGATT 28CAGACGTGTGCTCTTCCGATCTCC 29 ACCGAAAA ATCCAAGGGAGAGTGAGA CD138AATGGCAAAGGA 30 CAGACGTGTGCTCTTCCGATCTGC 31 AGGTGGAT AGACACCTTGGACATCCTCD38 AGATCTGAGCCA 32 CAGACGTGTGCTCTTCCGATCTTG 33 GTCGCTGTGTGCAGAGCTGAAGATTTT CD24 AAAAGTGGGCTT 34 CAGACGTGTGCTCTTCCGATCTTT 35GATTCTGC TTGTTCGCATGGTCACAC CD10 ATATTCCTTTGG 36CAGACGTGTGCTCTTCCGATCTTC 37 GCCTCTGC AAGTTTGGGTCTGTGCTG CD95CCCCCGAAAATG 38 CAGACGTGTGCTCTTCCGATCTTG 39 TTCAATAA CTCTTGTCATACCCCCACD21 TAGCTTCCTCCT 40 CAGACGTGTGCTCTTCCGATCTTT 41 CTGGTGGTTGCCTTTCCATAATCACTCA CXCR3 CTGGCTCTCCCC 42 CAGACGTGTGCTCTTCCGATCTGC 43AATATCCT TCTGAGGACTGCACCATT CD40 GTGGTGTTGGGG 44CAGACGTGTGCTCTTCCGATCTAT 45 TATGGTTT ACACAGATGCCCATTGCA CD69AGACAGGTCCTT 46 CAGACGTGTGCTCTTCCGATCTTG 47 TTCGATGG TGCAATATGTGATGTGGCCD1c TTGAGACAGGCA 48 CAGACGTGTGCTCTTCCGATCTTT 49 CATACAGCTTGCTTCCTCAATCTGTCCA IL10 CCCCAACCACTT 50 CAGACGTGTGCTCTTCCGATCTTT 51CATTCTTG CAATTCCTCTGGGAATGTT IL4R TGCCTAGAGGTG 52CAGACGTGTGCTCTTCCGATCTGT 53 CTCATTCA TGATGCTGGAGGCAGAAT IL21RAGCCTGGGTCAC 54 CAGACGTGTGCTCTTCCGATCTAG 55 AGATCAAG GTAGGAGGGTGGATGGAGIL6R CCAGCACCAGGG 56 CAGACGTGTGCTCTTCCGATCTAG 57 AGTTTCTAGAAAGGATTGGAACAGCA CXCL12 GGGTTTCAGGTT 58 CAGACGTGTGCTCTTCCGATCTTT 59CCAATCAG TGTAACTTTTTGCAAGGCA CCL3 GTGAGGAGTGGG 60CAGACGTGTGCTCTTCCGATCTAG 61 TCCAGAAA TGGGGAGGAGCAGGAG CCL14 CCATTCCCTTCT62 CAGACGTGTGCTCTTCCGATCTTA 63 TCCTCCTC CCTACAAGATCCCGCGTC CCL20TTGGACATAGCC 64 CAGACGTGTGCTCTTCCGATCTTG 65 CAAGAACA TGCCTCACTGGACTTGTCCCL18 ACCTGAAGCTGA 66 CAGACGTGTGCTCTTCCGATCTCT 67 ATGCCTGAGGAGGCCACCTCTTCTAA TCL1A GGTAAACACGCC 68 CAGACGTGTGCTCTTCCGATCTCA 69TGCAAAC GGACTCAGAAGCCTCTGG TACI CAACAAAGCACA 70 CAGACGTGTGCTCTTCCGATCTTG71 GTGTTAAATGAA TGTCAGCTACTGCGGAAA AICDA TGAGCAGATCCA 72CAGACGTGTGCTCTTCCGATCTGA 73 CAGGAAAA AATGGAGTCTCAAAGCTTCA FCRL4TCCCAACTACGC 74 CAGACGTGTGCTCTTCCGATCTGA 75 TGATTTGA CCAAAAGGAATGTGTGGGBCL2 TGCAAGAGTGAC 76 CAGACGTGTGCTCTTCCGATCTTC 77 AGTGGATTGAACCAAGGTTTGCTTTTGT FASLG AGAGGCTGAAAG 78 CAGACGTGTGCTCTTCCGATCTAA 79AGGCCAAT TATGGGTTGCATTTGGTCA BCL6 AAATCTGCAGAA 80CAGACGTGTGCTCTTCCGATCTAG 81 GGAAAAATGTG TTTTCAATGATGGGCGAG AURKBGCTCAAGGGAGA 82 CAGACGTGTGCTCTTCCGATCTGA 83 GCTGAAGA CTACCTGCCCCCAGAGATCD81 GTGGCGTGTATG 84 CAGACGTGTGCTCTTCCGATCTCA 85 AGTGGAGACTCGCCCAGAGACTCAG CD80 GCACATCTCATG 86 CAGACGTGTGCTCTTCCGATCTGC 87GCAGCTAA TTCACAAACCTTGCTCCT CD23a ACATTTTCTGCC 88CAGACGTGTGCTCTTCCGATCTAA 89 ACCCAAAC CAGCACCCTCTCCAGATG CD44GCCTGGTAGAAT 90 CAGACGTGTGCTCTTCCGATCTTT 91 TGGCTTTTCTTGTAGCCAACATTCATTCAA LEF1 CAATTGGCAGCC 92 CAGACGTGTGCTCTTCCGATCTGT 93CTATTTCA TCAGCAGACTGGTTTGCA CXCR5 CCGTGAGGATGT 94CAGACGTGTGCTCTTCCGATCTAC 95 CACTCAGA GAGGAAGCCCTAAGACGT PRKCBTTGAGCCTGGGG 96 CAGACGTGTGCTCTTCCGATCTGT 97 TGTAAGAC CTTCCAGGATTCACGGTGPRKCD GAGCACCTCCTG 98 CAGACGTGTGCTCTTCCGATCTTA 99 GAAGATTGAGCACCAGTGGGACTGTG CD20 TAGGAGCAGGCC 100 CAGACGTGTGCTCTTCCGATCTGA 101TGAGAAAA TTCCTCTCCAAACCCATG CD30 TGTTTTGGGGAA 102CAGACGTGTGCTCTTCCGATCTCT 103 AGTTGGAG GTTTGCCCAGTGTTTGTG CD30LTGCAACCCAACT 104 CAGACGTGTGCTCTTCCGATCTTT 105 GTGTGTTATCACCAACTGTTCTCTGAGC BAFFR GCCCTGAGCAAC 106 CAGACGTGTGCTCTTCCGATCTTT 107AATAGCAG CAGCTCTTCACTCCAGCA CMRF-35H AGGAAAAGATGT 108CAGACGTGTGCTCTTCCGATCTGG 109 GGCTCACG AGTTGGGGAGAACTGTCA PRDM1TCGAATAATCCA 110 CAGACGTGTGCTCTTCCGATCTAC 111 GGGAAACCCAAAGCATCACGTTGACA HLA-DRA GGCTTTACAAAG 112 CAGACGTGTGCTCTTCCGATCTTA 113CTGGCAAT TGCCTCTTCGATTGCTCC GNA12 CCTTGAGTGTGT 114CAGACGTGTGCTCTTCCGATCTCC 115 CTGCGTGT ACAGAATTGGGTTCCAAG RGS1AACTGGGAAGGC 116 CAGACGTGTGCTCTTCCGATCTTG 117 CAGGTAACTTTTCAAATTGCCATTGC CD5 CTTTCTCCACGC 118 CAGACGTGTGCTCTTCCGATCTAC 119CATTTGAT TAGGATATGGGGTGGGCT CD22 GGGATCTGCTCG 120CAGACGTGTGCTCTTCCGATCTGT 121 TCATCATT TTCTGCCTCTGAGGGAAA PIK3CDGCGTGCGCGTTA 122 CAGACGTGTGCTCTTCCGATCTTG 123 TTTATTTATCTGGGGAAGGCAAGTTA DOCK8 GCAGTCAGCCAG 124 CAGACGTGTGCTCTTCCGATCTTT 125AAATCACA TTCTCCTCTCTGGGACCA CD11b TGAAAAGTCTCC 126CAGACGTGTGCTCTTCCGATCTCC 127 CTTTCCAGA TTCAGACAGATTCCAGGC FCGR2BGGAGAGGAGAGA 128 CAGACGTGTGCTCTTCCGATCTGA 129 TGGGGATTGTGAGTGCCCCTTTTCTT CD72 CTCATGCCAACA 130 CAGACGTGTGCTCTTCCGATCTTG 131AGAACCTG ACCCACACCTGACACTTC BCL11B TCGTGGAACACA 132CAGACGTGTGCTCTTCCGATCTTT 133 GGCAAAC GCATTTGTACTGGCAAGG CD86TCAAGGCAACCA 134 CAGACGTGTGCTCTTCCGATCTAC 135 GAGGAAACTAAGGGATGGGGCAGTCT TBX21 ACCTTTTCGTTG 136 CAGACGTGTGCTCTTCCGATCTTC 137GCATGTGT AGGGAAAGGACTCACCTG FOXP1 ATGCTGAAGGCA 138CAGACGTGTGCTCTTCCGATCTCT 139 TTTCTTGG GTGAGCATGGTGCTTCAT MCL1GAGGGGAGTGGT 140 CAGACGTGTGCTCTTCCGATCTCA 141 GGGTTTATAAAGGGAAAGGGAGGATT IFNB1 AGGGGAAAACTC 142 CAGACGTGTGCTCTTCCGATCTTC 143ATGAGCAG ACTGTGCCTGGACCATAG BLNK TTGGGCAGAAAG 144CAGACGTGTGCTCTTCCGATCTCA 145 AAAAATGG AAAGATTCCACCAGACTGAA CD40LGCCTCCCCCAGTC 146 CAGACGTGTGCTCTTCCGATCTGA 147 TCTCTTCTGTCAGGCCGTTGCTAGTC IGBP1 GGCTGATCTTCC 148 CAGACGTGTGCTCTTCCGATCTAC 149CACAACAC GAGGGCAAAGATGCTAAA IRF4 ATTCCCGTGTTG 150CAGACGTGTGCTCTTCCGATCTAG 151 CTTCAAAC AACTGCCAGCAGGTAGGA CD79aCACTTCCCTGGG 152 CAGACGTGTGCTCTTCCGATCTCT 153 ACATTCTCCACTCTTCTCCAGGCCAG LTA TGATGTCTGTCT 154 CAGACGTGTGCTCTTCCGATCTCC 155GGCTGAGG ACACACAGAGGAAGAGCA HDAC5 CCAGCCTGTAGG 156CAGACGTGTGCTCTTCCGATCTCT 157 AAACCAAA CCTTCTATCTCCAGGGCC RAG1GGATGCAGGTGG 158 CAGACGTGTGCTCTTCCGATCTCA 159 TTTTTGATTTGTACCCATTTTACATTTTCTT RAG2 CAAACCTTAAAC 160 CAGACGTGTGCTCTTCCGATCTAT161 ACCCAGAAGC AACAATTCGGCAGTTGGC CD1d GAACCAGTTTCC 162CAGACGTGTGCTCTTCCGATCTAA 163 TCCTGTGC GATGTGGAGGCTGTTGCT TGFB1GACTGCGGATCT 164 CAGACGTGTGCTCTTCCGATCTTC 165 CTGTGTCATGCACTATTCCTTTGCCC CD9 TCAGTATGATCT 166 CAGACGTGTGCTCTTCCGATCTTA 167TGTGCTGTGCT CCCATGAAGATTGGTGGG CD11c CACAGCATGAGA 168CAGACGTGTGCTCTTCCGATCTTC 169 GGCTCTGT TCAGTTCCGATTTCCCAG FOXP3TCAGGATCTGAG 170 CAGACGTGTGCTCTTCCGATCTTC 171 GTCCCAACACCTGTGTATCTCACGCA LAG3 AGAGCTGTCTAG 172 CAGACGTGTGCTCTTCCGATCTTG 173CCCAGGTG GTGTCCTTTCTCTGCTCC CD73 CTTAACGTGGGA 174CAGACGTGTGCTCTTCCGATCTGT 175 GTGGAACC GTGCAAATGGCAGCTAGA CD70TCTCAGCTTCCA 176 CAGACGTGTGCTCTTCCGATCTTC 177 CCAAGGTTACTGGGACACTTTTGCCT CCR7 CAGGGGAGAGTG 178 CAGACGTGTGCTCTTCCGATCTGA 179TGGTGTTT CATGCACTCAGCTCTTGG CD45RA TGCATAGTTCCC 180CAGACGTGTGCTCTTCCGATCTTA 181 ATGTTAAATCC CCAGGAATGGATGTCGCT PDCD1ACATCCTACGGT 182 CAGACGTGTGCTCTTCCGATCTGC 183 CCCAAGGT AGAAGTGCAGGCACCTAMYC TGCATGATCAAA 184 CAGACGTGTGCTCTTCCGATCTTT 185 TGCAACCTGGACTTTGGGCATAAAAGA CD25 AAATCACGGCAG 186 CAGACGTGTGCTCTTCCGATCTCT 187TTTTCAGC CATCTGTGCACTCTCCCC FCAMR GTGGGAAGAGAA 188CAGACGTGTGCTCTTCCGATCTTC 189 GCTGATGC AAGCATTATCCACGTCCA CCND2TGTGATGCCATA 190 CAGACGTGTGCTCTTCCGATCTTC 191 TCAAGTCCAAGTGTATGCGAAAAGGTTTTT MKI67 AGCCTCTCTTGG 192 CAGACGTGTGCTCTTCCGATCTGT193 GCTTTCTT TTTCCCTGCCTGGAACTT CCND3 CTTTGCTGCTGA 194CAGACGTGTGCTCTTCCGATCTAC 195 AGGCTCAT AAGTGGTGGTAACCCTGG IL12ATGCTTCCTAAAA 196 CAGACGTGTGCTCTTCCGATCTGA 197 AGCGAGGTACTAGGGAGGGGGAAAGA IFNG GCAGCCAACCTA 198 CAGACGTGTGCTCTTCCGATCTAT 199AGCAAGAT CCAGTTACTGCCGGTTTG TNFA GAATGCTGCAGG 200CAGACGTGTGCTCTTCCGATCTAC 201 ACTTGAGA TTCCTTGAGACACGGAGC IL2ACCCAGGGACTT 202 CAGACGTGTGCTCTTCCGATCTGC 203 AATCAGCATGATGAGACAGCAACCATT IL4 GACATCTTTGCT 204 CAGACGTGTGCTCTTCCGATCTAT 205GCCTCCA GAGAAGGACACTCGCTGC IL6 TTAAGGAGTTCC 206 CAGACGTGTGCTCTTCCGATCTTC207 TGCAGTCCA CACTGGGCACAGAACTTA BAFF TCCTTCGCTTTG 208CAGACGTGTGCTCTTCCGATCTAG 209 CTTGTCTT GTGGAAAAATAGATGCCAGTC IGHECCCGGAAGTCTA 210 CAGACGTGTGCTCTTCCGATCTAG 211 TGCGTTT GACATCTCGGTGCAGTGIGHD TGTGTGAGGTGT 212 CAGACGTGTGCTCTTCCGATCTAG 213 CTGGCTTCGAGCACCACGTTCTGG IGHM CCCGGAGAAGTA 214 CAGACGTGTGCTCTTCCGATCTGT 215TGTGACCA ACTTCGCCCACAGCATC IGHA CTGAACGAGCTG 216CAGACGTGTGCTCTTCCGATCTAG 217 GTGACG TACCTGACTTGGGCATCC IGHG1CAAGGGCCCATC 218 CAGACGTGTGCTCTTCCGATCTTT 219 GGTCTTGTGACAAAACTCACACATGC IGHG4 CAAGGGCCCATC 220 CAGACGTGTGCTCTTCCGATCTCA 221GGTCTT AATATGGTCCCCCATGC IGHG2 CAAGGGCCCATC 222 CAGACGTGTGCTCTTCCGATCTGC223 GGTCTT AAATGTTGTGTCGAGTGC IGHG3 CAAGGGCCCATC 224CAGACGTGTGCTCTTCCGATCTAC 225 GGTCTT CCCACTTGGTGACACAAC TLR1 CCATTCCGCAGT226 CAGACGTGTGCTCTTCCGATCTAA 227 ACTCCATT GGAAAAGAGCAAACGTGG TLR2TTGGTTGACTTC 228 CAGACGTGTGCTCTTCCGATCTGG 229 ATGGATGCAAACAGCACAAATGAACTTAA TLR3 CATCATGCAGTT 230 CAGACGTGTGCTCTTCCGATCTAT 231CAACAAGC GCACTCTGTTTGCGAAGA TLR4 GGGTGTGTTTCC 232CAGACGTGTGCTCTTCCGATCTTT 233 ATGTCTCA GAAAGTGTGTGTGTCCGC TLR5TCAGGCTGTTGC 234 CAGACGTGTGCTCTTCCGATCTGT 235 ATGAAGAAATGCCCTTGCTGGACCTA TLR6 ATGCGCAGTAAA 236 CAGACGTGTGCTCTTCCGATCTTA 237AACTCGTG CAGTTCCACGCTGAGCTG TLR7 GCCTGTACTTTC 238CAGACGTGTGCTCTTCCGATCTAA 239 AGCTGGGTA GGTGTTTGTGCCATTTGG TLR8GGTGAGCTCTGA 240 CAGACGTGTGCTCTTCCGATCTTA 241 TTGCTTCATCAGGAGGCAGGGATCAC TLR9 GACCGGGTCAGT 242 CAGACGTGTGCTCTTCCGATCTGG 243GGTCTCT TGATCCTGAGCCCTGAC TLR10 TGCAGTGAGCTG 244CAGACGTGTGCTCTTCCGATCTAT 245 AGATCGAG GGAAAACATCCTCATGGC GAPDH CACATGGCCUCC 246 CAGACGTGTGCTCTTCCGATCTCA 247 AAGGAGUAAGCAAGAGCACAAGAGGAA

TABLE 19 SEQ ID Nested Primer with Common SEQ ID Gene Outer Primer NO:5′ Flanking Sequence NO: CD19 GCAGGGTCCCAG 248 CAGACGTGTGCTCTTCCGATCTCCA249 TCCTATG ATCATGAGGAAGATCCA CD20 TAGGAGCAGGCC 250CAGACGTGTGCTCTTCCGATCTGAT 251 TGAGAAAA TCCTCTCCAAACCCATG BAFFTCCTTCGCTTTG 252 CAGACGTGTGCTCTTCCGATCTAGG 253 CTTGTCTTTGGAAAAATAGATGCCAGTC TCL1A GGTAAACACGCC 254 CAGACGTGTGCTCTTCCGATCTCAG255 TGCAAAC GACTCAGAAGCCTCTGG TACI CAACAAAGCACA 256CAGACGTGTGCTCTTCCGATCTTGT 257 GTGTTAAATGAA GTCAGCTACTGCGGAAA IGHDTGTGTGAGGTGT 258 CAGACGTGTGCTCTTCCGATCTAGG 259 CTGGCTTC AGCACCACGTTCTGGIGHM CCCGGAGAAGTA 260 CAGACGTGTGCTCTTCCGATCTGTA 261 TGTGACCACTTCGCCCACAGCATC CD27 TCCAGGAGGATT 262 CAGACGTGTGCTCTTCCGATCTCCA 263ACCGAAAA TCCAAGGGAGAGTGAGA CD38 AGATCTGAGCCA 264CAGACGTGTGCTCTTCCGATCTTGG 265 GTCGCTGT TGCAGAGCTGAAGATTTT CD24AAAAGTGGGCTT 266 CAGACGTGTGCTCTTCCGATCTTTT 267 GATTCTGCTGTTCGCATGGTCACAC AICDA TGAGCAGATCCA 268 CAGACGTGTGCTCTTCCGATCTGAA 269CAGGAAAA ATGGAGTCTCAAAGCTTCA CD95 CCCCCGAAAATG 270CAGACGTGTGCTCTTCCGATCTTGC 271 TTCAATAA TCTTGTCATACCCCCA CD10ATATTCCTTTGG 272 CAGACGTGTGCTCTTCCGATCTTCA 273 GCCTCTGCAGTTTGGGTCTGTGCTG IL10 CCCCAACCACTT 274 CAGACGTGTGCTCTTCCGATCTTTC 275CATTCTTG AATTCCTCTGGGAATGTT CD138 AATGGCAAAGGA 276CAGACGTGTGCTCTTCCGATCTGCA 277 AGGTGGAT GACACCTTGGACATCCT CD45RATGCATAGTTCCC 278 CAGACGTGTGCTCTTCCGATCTTAC 279 ATGTTAAATCCCAGGAATGGATGTCGCT BCL6 AAATCTGCAGAA 280 CAGACGTGTGCTCTTCCGATCTAGT 281GGAAAAATGTG TTTCAATGATGGGCGAG PRDM1 TCGAATAATCCA 282CAGACGTGTGCTCTTCCGATCTACC 283 GGGAAACC AAAGCATCACGTTGACA CXCR3CTGGCTCTCCCC 284 CAGACGTGTGCTCTTCCGATCTGCT 285 AATATCCTCTGAGGACTGCACCATT IFNG GCAGCCAACCTA 286 CAGACGTGTGCTCTTCCGATCTATC 287AGCAAGAT CAGTTACTGCCGGTTTG IL4R TGCCTAGAGGTG 288CAGACGTGTGCTCTTCCGATCTGTT 289 CTCATTCA GATGCTGGAGGCAGAAT IL4GACATCTTTGCT 290 CAGACGTGTGCTCTTCCGATCTATG 291 GCCTCCA AGAAGGACACTCGCTGCCCL20 TTGGACATAGCC 292 CAGACGTGTGCTCTTCCGATCTTGT 293 CAAGAACAGCCTCACTGGACTTGTC CD25 AAATCACGGCAG 294 CAGACGTGTGCTCTTCCGATCTCTC 295TTTTCAGC ATCTGTGCACTCTCCCC FOXP1 ATGCTGAAGGCA 296CAGACGTGTGCTCTTCCGATCTCTG 297 TTTCTTGG TGAGCATGGTGCTTCAT TGFB1GACTGCGGATCT 298 CAGACGTGTGCTCTTCCGATCTTCT 299 CTGTGTCAGCACTATTCCTTTGCCC CXCR5 CCGTGAGGATGT 300 CAGACGTGTGCTCTTCCGATCTACG 301CACTCAGA AGGAAGCCCTAAGACGT CD69 AGACAGGTCCTT 302CAGACGTGTGCTCTTCCGATCTTGT 303 TTCGATGG GCAATATGTGATGTGGC CD30TGTTTTGGGGAA 304 CAGACGTGTGCTCTTCCGATCTCTG 305 AGTTGGAGTTTGCCCAGTGTTTGTG PDCD1 ACATCCTACGGT 306 CAGACGTGTGCTCTTCCGATCTGCA 307CCCAAGGT GAAGTGCAGGCACCTA LAG3 AGAGCTGTCTAG 308CAGACGTGTGCTCTTCCGATCTTGG 309 CCCAGGTG TGTCCTTTCTCTGCTCC PAX5TGACGTGTGTTG 310 CAGACGTGTGCTCTTCCGATCTACT 311 CTTTTGTGTGGGAGAAAACAGGGGT TNFRSF17 GCTTTCCACTCC 312 CAGACGTGTGCTCTTCCGATCTTGC313 CAGCTATG TTTGAGTGCTACGGAGA RASD1 GGGGGAGGGATG 314CAGACGTGTGCTCTTCCGATCTATC 315 TGAAGTTA TTGTCTGTGATTCCGCG AMPD1ACAGATGACCCA 316 CAGACGTGTGCTCTTCCGATCTGAG 317 ATGCAATTCCACCTGTGATATGTGCG OSBPL5 AGACCGATGCAC 318 CAGACGTGTGCTCTTCCGATCTCTT 319AGTCTTCC CACGTCTGGCCTCAGTC CD56 GGAGCACTCAAG 320CAGACGTGTGCTCTTCCGATCTTTT 321 TGTGACGA TCTATGGAGCCTTCCGA IGFBP7CATCCAATTCCC 322 CAGACGTGTGCTCTTCCGATCTGGT 323 AAGGACAGGAAGGTGCCGAGCTATA KIR2DS5 GCTCTTCCTCAA 324 CAGACGTGTGCTCTTCCGATCTCAC 325ACCACGAA ACTCCTTTGCTTAGCCC KIR2DS2 TCCTCACACCAC 326CAGACGTGTGCTCTTCCGATCTCAC 327 GAATCTGA TCCTTTGCTTAGCCCAC RAB4BCCAGCTCACCTG 328 CAGACGTGTGCTCTTCCGATCTGAA 329 TTCTCCAG TCCCGTACCTGCTGCTCD14 CTAAAGGACTGC 330 CAGACGTGTGCTCTTCCGATCTATA 331 CAGCCAAGACCTGACACTGGACGCG S100A12 CACATTCCTGTG 332 CAGACGTGTGCTCTTCCGATCTATA 333CATTGAGG CTCAGTTCGGAAGGGGC CCR2variantB GAAGGAGGGAGA 334CAGACGTGTGCTCTTCCGATCTACT 335 CATGAGCA GGTCCTTAGCCCCATCT CD62LTCAGTTGGCTGA 336 CAGACGTGTGCTCTTCCGATCTTTA 337 CTTCCACAGTTTGGGGGTTTTGCTG CD16 TCTTGGCCAGGG 338 CAGACGTGTGCTCTTCCGATCTGTC 339TAGTAAGAA AGTTCCAATGAGGTGGG CX3CR1 CGTCCAGACCTT 340CAGACGTGTGCTCTTCCGATCTCCA 341 GTTCACAC CAAATAGTGCTCGCTTTC CD1bTAGAGGGCCAGG 342 CAGACGTGTGCTCTTCCGATCTTTG 343 ACATCATCCTCCTTTTGCTATGCCT FOXQ1 TGCTATTGACCG 344 CAGACGTGTGCTCTTCCGATCTGCA 345ATGCTTCA ACGGGCTACAGCTTTAT CD209 GCTCTTGTTCTT 346CAGACGTGTGCTCTTCCGATCTGAG 347 GCCGTTTT TCCCTCAGTGGAGCAAG CD1eCACAAGCACATT 348 CAGACGTGTGCTCTTCCGATCTATT 349 CATCTCTTCCCAGGGCCAGCTTCATAA CCL17 TACTTCAAGGGA 350 CAGACGTGTGCTCTTCCGATCTTTT 351GCCATTCC GTAACTGTGCAGGGCAG DTNA AGCAACGTGGAG 352CAGACGTGTGCTCTTCCGATCTCTC 353 TCAGTCTGT ACCTTCTCTTGCCTTGG CLEC4CTTATTTTCTGGG 354 CAGACGTGTGCTCTTCCGATCTCAT 355 GCTGTCAGATCTGGCACTCAGGTGAA ZBTB16 TGATCAAGCACC 356 CAGACGTGTGCTCTTCCGATCTTAC 357TGAGAACG CAGTGCACCATCTGCAC SLAMF1 TGCAAAACCCAG 358CAGACGTGTGCTCTTCCGATCTGTT 359 AAGCTAAAA CTGTGCAAATGGCATTC CD3DAGAGCTGTGTGG 360 CAGACGTGTGCTCTTCCGATCTGGA 361 AGCTGGATGTCTTCTGCTTTGCTGG CD3E GCCCTCTTGCCA 362 CAGACGTGTGCTCTTCCGATCTGCA 363GGATATTT TGTAAGTTGTCCCCCAT CD8A CTGGCCTCTGCT 364CAGACGTGTGCTCTTCCGATCTATG 365 CAACTAGC GTACAAGCAATGCCTGC CD8BCAGCCTCAAGGG 366 CAGACGTGTGCTCTTCCGATCTTGC 367 GAAGGTATTTAACCCATGGATCCTG PRF1 CCCTGCAGTCAC 368 CAGACGTGTGCTCTTCCGATCTTCA 369AGCTACAC GGGCTGGTCTTTTAGGA EOMES TGGGATAATGTA 370CAGACGTGTGCTCTTCCGATCTCAT 371 AAACTGGTGCT CCCCATGATATTTGGGA CD4AGCTAGCCTGAG 372 CAGACGTGTGCTCTTCCGATCTTCC 373 AGGGAACCTCCAGACCATTCAGGAC THPOK GGCTCTGCCTTG 374 CAGACGTGTGCTCTTCCGATCTCTC 375CACTATTT TTCCTCCCTTCCATGC RUNX3 TAAGGCCCAAAG 376CAGACGTGTGCTCTTCCGATCTTAG 377 TGGGTACA GAAGCACGAGGAAAGGA CD45ROACCCTCTCTCCC 378 CAGACGTGTGCTCTTCCGATCTTAG 379 TCCCTTTCTTGGCTATGCTGGCATG CD44 GCCTGGTAGAAT 380 CAGACGTGTGCTCTTCCGATCTTTT 381TGGCTTTTC TGTAGCCAACATTCATTCAA CCR7 CAGGGGAGAGTG 382CAGACGTGTGCTCTTCCGATCTACT 383 TGGTGTTT CAGCTCTTGGCTCCACT TXKACATCAAGCTCC 384 CAGACGTGTGCTCTTCCGATCTTTT 385 ATTGTTTCGGCCTGCACTCTTTGTAGG MBD2 GCCTGGCACGTA 386 CAGACGTGTGCTCTTCCGATCTAGG 387ATAGCTTG AAAGAAATGCCCTTGGT IFNGR1 GAGGATGTGTGG 388CAGACGTGTGCTCTTCCGATCTGGT 389 CATTTTCA TCCTAGGTGAGCAGGTG IL12RB2AGCAGGCTGTAC 390 CAGACGTGTGCTCTTCCGATCTGAC 391 ACAGCAGAACTAGGCACATTGGCTG IL33R ACTGTGCCCTCA 392 CAGACGTGTGCTCTTCCGATCTAAC 393TCCAGAAC GACGCCAAGGTGATACT CCR4 TGGTGAAATGCA 394CAGACGTGTGCTCTTCCGATCTTCA 395 GAGTCAATG GGAGGAAGGCTTACACC CRTH2TGAATTTTGCTT 396 CAGACGTGTGCTCTTCCGATCTTGT 397 GGTGGATGCAGTGGAAGAAGCAGATG IL5 CAGTGAGAATGA 398 CAGACGTGTGCTCTTCCGATCTGAA 399GGGCCAAG TGAGGGCCAAGAAAGAG IL17A AAAATGAAACCC 400CAGACGTGTGCTCTTCCGATCTTCC 401 TCCCCAAA TTTGGAGATTAAGGCCC IL17FCTGCATCAATGC 402 CAGACGTGTGCTCTTCCGATCTCCA 403 TCAAGGAAAGGCTGCTCTGTTTCTT IL21 AAATCAAGCTCC 404 CAGACGTGTGCTCTTCCGATCTTGT 405CAAGGTCA GAATGACTTGGTCCCTG IL22 ATGCCCCAAAGC 406CAGACGTGTGCTCTTCCGATCTCAA 407 GATTTTT AGGAAACCAATGCCACT IL23RTCCCTCATTGAA 408 CAGACGTGTGCTCTTCCGATCTTAG 409 AGATGCAAAATCATTAGGCCACGCG RORA TGCAAGCCATTT 410 CAGACGTGTGCTCTTCCGATCTCCT 411ATGGGAAT TGGGTTTTCTTTTCAATTC RORC ATTTCCATGGTG 412CAGACGTGTGCTCTTCCGATCTAGA 413 CTCCAGTC GAAGCAGAAGTCGCTCG OX40LCTGCTGGCCCTG 414 CAGACGTGTGCTCTTCCGATCTCTC 415 TACCTG CACCCTGGCCAAGATICOS TTCAGCTGACTT 416 CAGACGTGTGCTCTTCCGATCTGGA 417 GGACAACCTCAACCTGACTGGCTTTG SH2D1A  GGGTGTTGGTGA 418 CAGACGTGTGCTCTTCCGATCTTTT 419ACTTGGTT AATATGGATGCCGTGGG CCR2variantA GTTGCCCAGTGT 420CAGACGTGTGCTCTTCCGATCTAAC 421 GTTTCTGA CAGGCAACTTGGGAACT TLR1CCATTCCGCAGT 422 CAGACGTGTGCTCTTCCGATCTAAG 423 ACTCCATTGAAAAGAGCAAACGTGG TLR2 TTGGTTGACTTC 424 CAGACGTGTGCTCTTCCGATCTGGA 425ATGGATGC AACAGCACAAATGAACTTAA TLR3 CATCATGCAGTT 426CAGACGTGTGCTCTTCCGATCTATG 427 CAACAAGC CACTCTGTTTGCGAAGA TLR4GGGTGTGTTTCC 428 CAGACGTGTGCTCTTCCGATCTTTG 429 ATGTCTCAAAAGTGTGTGTGTCCGC TLR5 TCAGGCTGTTGC 430 CAGACGTGTGCTCTTCCGATCTGTA 431ATGAAGAA TGCCCTTGCTGGACCTA TLR6 ATGCGCAGTAAA 432CAGACGTGTGCTCTTCCGATCTTAC 433 AACTCGTG AGTTCCACGCTGAGCTG TLR7GCCTGTACTTTC 434 CAGACGTGTGCTCTTCCGATCTAAG 435 AGCTGGGTAGTGTTTGTGCCATTTGG TLR8 GGTGAGCTCTGA 436 CAGACGTGTGCTCTTCCGATCTTAT 437TTGCTTCA CAGGAGGCAGGGATCAC TLR9 GACCGGGTCAGT 438CAGACGTGTGCTCTTCCGATCTGGT 439 GGTCTCT GATCCTGAGCCCTGAC TLR10TGCAGTGAGCTG 440 CAGACGTGTGCTCTTCCGATCTATG 441 AGATCGAGGAAAACATCCTCATGGC GAPDH CACATGGCCUCC 442 CAGACGTGTGCTCTTCCGATCTCAG 443AAGGAGUAA CAAGAGCACAAGAGGAAStudying Diversity of Response of Human T Cells to In Vitro Stimulus

When examining the gene expression pattern of a bulk sample, theobserved pattern was contributed by both the sample's cell compositionand the expression level of each gene in each cell type or subtype.These two effects cannot be deconvoluted by bulk analysis but only withlarge-scale single cell analysis. To illustrate, we utilized ourplatform to study the variability of response of human T cells to an invitro stimulus.

We purified CD3+ T cells by negative selection from a blood donor andstimulated them with anti-CD28/anti-CD3 beads for 6 hours, and performedexperiments with the stimulated and a separate aliquot of unstimulatedcells. We designed a panel of 93 genes (Table 20) that encompassedsurface proteins, cytokines, chemokines, and effector moleculesexpressed by the different T cell subsets. A total of 3517 and 1478single cells were analyzed for the stimulated and unstimulated samples,respectively.

TABLE 20 SEQ ID Nested Primer with Common SEQ ID Gene Outer Primer NO:5′ Flanking Sequence NO: GAPDH GACTTCAACAGC 444CAGACGTGTGCTCTTCCGATCTGCC 445 GACACCCA CTCAACGACCACTTTGT CD3DGAAAACGCATCC 446 CAGACGTGTGCTCTTCCGATCTTGA 447 TGGACCCATGTCATTGCCACTCTGCT CD3E AAGTTGTCCCCC 448 CAGACGTGTGCTCTTCCGATCTCTG 449ATCCCAAA GGGATGGACTGGGTAAAT CD8A ACTGCTGTCCCA 450CAGACGTGTGCTCTTCCGATCTATG 451 AACATGCA CCTGCCCATTGGAGAGAA CD8BCCACCATCTTTG 452 CAGACGTGTGCTCTTCCGATCTGCT 453 CAGGTTGCGTCCAGTTCCCAGAAGG CD4 CTGGGAGAGGGG 454 CAGACGTGTGCTCTTCCGATCTACC 455GTAGCTAG ACTTCCCTCAGTCCCAA FOXP3 ACAGAAGCAGCG 456CAGACGTGTGCTCTTCCGATCTGGG 457 TCAGTACC TCTCTTGAGTCCCGTG CCR7GGGGAGAGTGTG 458 CAGACGTGTGCTCTTCCGATCTCTC 459 GTGTTTCCTTGGCTCCACTGGGATG CD5 ATCAATGGTCCA 460 CAGACGTGTGCTCTTCCGATCTAGG 461AGCCGCAT TCACAGATCTTCCCCCG IL32 CTTTCCAGTCCT 462CAGACGTGTGCTCTTCCGATCTTGC 463 ACGGAGCC TCTGAACCCCAATCCTC CD28ACCATCACAGGC 464 CAGACGTGTGCTCTTCCGATCTTGT 465 ATGTTCCTAGATGACCTGGCTTGCC SELL GCATCTCATGAG 466 CAGACGTGTGCTCTTCCGATCTCCT 467TGCCAAGC GCCCCCAGACCTTTTATC CD27 TGCAGAGCCTTG 468CAGACGTGTGCTCTTCCGATCTCGT 469 TCGTTACA GACAGAGTGCCTTTTCG GZMBAGGTGAAGATGA 470 CAGACGTGTGCTCTTCCGATCTAGG 471 CAGTGCAGGCCCTCTTGTGTGTAACA GZMA GGAACCATGTGC 472 CAGACGTGTGCTCTTCCGATCTCCT 473CAAGTTGC TTGTTGTGCGAGGGTGT GZMH AGTGTTGCTGAC 474CAGACGTGTGCTCTTCCGATCTCCA 475 AGTGCAGA AAGAAGACACAGACCGGT GZMKTTGCCACAAAGC 476 CAGACGTGTGCTCTTCCGATCTAAA 477 CTGGAATCGCAACCTTGTCCCGCCT PRF1 GGAGTCCAGCGA 478 CAGACGTGTGCTCTTCCGATCTCAT 479ATGACGTC GGCCACGTTGTCATTGT NKG2D CAACACCCAGGG 480CAGACGTGTGCTCTTCCGATCTCCA 481 GATCAGTG CCCTCCACAGGAAATTG LAG3AGCTGTACCAGG 482 CAGACGTGTGCTCTTCCGATCTCTT 483 GGGAGAGTGGAGAAGACAGTGGCGA CD160 GGAAGACAGCCA 484 CAGACGTGTGCTCTTCCGATCTTTG 485GATCCAGTG TGCAGACCAAGAGCACC CD244 GGGCTGAGAATG 486CAGACGTGTGCTCTTCCGATCTGGA 487 AGGCAGTT AAGCGACAAGGGTGAAC EOMESACTTAACAGCTG 488 CAGACGTGTGCTCTTCCGATCTACT 489 CAGGGGCAACTTGAACCGTGTTTAAGG TBX21 TTATAACCATCA 490 CAGACGTGTGCTCTTCCGATCTAGA491 GCCCGCCA AAAGGGGCTGGAAAGGG PRDM1 ACCAAAGCATCA 492CAGACGTGTGCTCTTCCGATCTACA 493 CGTTGACAT TGTGAATGTTGAGCCCA IRF4CTCTTCAGCATC 494 CAGACGTGTGCTCTTCCGATCTGCC 495 CCCCGTACCCCAAATGAAAGCTTGA ZNF683 GGAGAGCGTCCA 496 CAGACGTGTGCTCTTCCGATCTATC 497TTCCAGTG CACCTGAAGCTGCACC ZBED2 AATGTACCAGCC 498CAGACGTGTGCTCTTCCGATCTGGT 499 AGTCAGCG TTTGGTGGAGCTGACGA CD30TTTACTCATCGG 500 CAGACGTGTGCTCTTCCGATCTTGT 501 GCAGCCACTTGCCCAGTGTTTGTGC CD69 GCTGTAGACAGG 502 CAGACGTGTGCTCTTCCGATCTAGT 503TCCTTTTCG GTTGGAAAATGTGCAATATGTG HLA-DRA GGGTCTGGTGGG 504CAGACGTGTGCTCTTCCGATCTGCC 505 CATCATTA TCTTCGATTGCTCCGTA CD38AGGTCAATGCCA 506 CAGACGTGTGCTCTTCCGATCTATC 507 GAGACGGAAGCATACCTTTATTGTGATCTATC TNFRSF9 TGGCATGTGAGT 508CAGACGTGTGCTCTTCCGATCTTTT 509 CATTGCTC TGATGTGAGGGGCGGAT MKI67TACTTTTTCGCC 510 CAGACGTGTGCTCTTCCGATCTTCC 511 TCCCAGGGTGCCCCACCAAGATCAT BIRC5 TGCCACGGCCTT 512 CAGACGTGTGCTCTTCCGATCTTTG 513TCCTTAAA TCTAAGTGCAACCGCCT FOSL1 CTCCTGACAGAA 514CAGACGTGTGCTCTTCCGATCTGGT 515 GGTGCCAC GATTGGACCAGGCCATT MCL1GACTGGCTACGT 516 CAGACGTGTGCTCTTCCGATCTTTT 517 AGTTCGGGGCTTAGAAGGATGGCGC MYC AGCTACGGAACT 518 CAGACGTGTGCTCTTCCGATCTCAA 519CTTGTGCG CCTTGGCTGAGTCTTGA TYMS TCAGTCTTTAGG 520CAGACGTGTGCTCTTCCGATCTATG 521 GGTTGGGC TGCATTTCAATCCCACGTAC CDCA7CCAGTCTAGTTT 522 CAGACGTGTGCTCTTCCGATCTATG 523 CTGGGCAGGTAAACCATTGCTGTGCCATT UHRF1 CCAGTTCTTCCT 524 CAGACGTGTGCTCTTCCGATCTCCA525 GACACCGG AAGTTTGCAGCCTATACC SAP30 ACCAACCAGACC 526CAGACGTGTGCTCTTCCGATCTTCA 527 AGGACTTA CTAGGAGACGTGGAATTG CX3CR1CACCCGTCCAGA 528 CAGACGTGTGCTCTTCCGATCTTGT 529 CCTTGTTTTTCCTCTTAACGTTAGACCAC BCL2 TGCAAGAGTGAC 530 CAGACGTGTGCTCTTCCGATCTGCT531 AGTGGATTG GATATTCTGCAACACTGTACA BCL6 TGTCCTCACGGT 532CAGACGTGTGCTCTTCCGATCTGTA 533 GCCTTTT GGCAGACACAGGGACTT FASLGCCTCAAGGGGGA 534 CAGACGTGTGCTCTTCCGATCTGCA 535 CTGTCTTTCTATCCTGAGCCATCGGT FAS ATTGCTGGTAGA 536 CAGACGTGTGCTCTTCCGATCTCCC 537GACCCCCA CCATTTCCCCGATGT CCL4 CCCAGCCAGCTG 538 CAGACGTGTGCTCTTCCGATCTTGG539 TGGTATTC AACTGAACTGAGCTGCT IFNG CTAGGCAGCCAA 540CAGACGTGTGCTCTTCCGATCTCCT 541 CCTAAGCA GCAATCTGAGCCAGTGC TNFAGTGGACCTTAG 542 CAGACGTGTGCTCTTCCGATCTGGC 543 GCCTTCCTTCAGACATGTTTTCCGTG IL2 TCACTTAAGACC 544 CAGACGTGTGCTCTTCCGATCTAAG 545CAGGGACTT CATCATCTCAACACTGACTT IL4 ACCATGAGAAGG 546CAGACGTGTGCTCTTCCGATCTCGG 547 ACACTCGC GCTTGAATTCCTGTCCT IL6CGGCAAATGTAG 548 CAGACGTGTGCTCTTCCGATCTGGA 549 CATGGGCAAGTGGCTATGCAGTTTG IL1A GGCATCCTCCAC 550 CAGACGTGTGCTCTTCCGATCTGCA 551AATAGCAGA TTTTGGTCCAAGTTGTGC IL1B CTTAAAGCCCGC 552CAGACGTGTGCTCTTCCGATCTACA 553 CTGACAGA TTCTGATGAGCAACCGC IL3ACAGACGACTTT 554 CAGACGTGTGCTCTTCCGATCTATT 555 GAGCCTCGTCACCTTTTCCTGCGGC IL13 GGAGCCAAGGGT 556 CAGACGTGTGCTCTTCCGATCTTGC 557TCAGAGAC TACCTCACTGGGGTCCT IL31 GGCCATCTCTTC 558CAGACGTGTGCTCTTCCGATCTGTG 559 CTTTCGGA TGGGAACTCTGCCGTG IL24CTCACCCCATCA 560 CAGACGTGTGCTCTTCCGATCTGCC 561 TCCCTTTCCCAGTGAGACTGTGTTGT IL26 TACTGACGGCAT 562 CAGACGTGTGCTCTTCCGATCTTGT 563GTTAGGTG GTGTGGAGTGGGATGTG LTA AGGCAGGGAGGG 564CAGACGTGTGCTCTTCCGATCTGGA 565 GACTATTT GAAACAGAGACAGGCCC IL5GCAGTGAGAATG 566 CAGACGTGTGCTCTTCCGATCTAGG 567 AGGGCCA CATACTGACACTTTGCCCSF2 AGCCAGTCCAGG 568 CAGACGTGTGCTCTTCCGATCTGGC 569 AGTGAGACCACACTGACCCTGATAC IL21 CCCAAGGTCAAG 570 CAGACGTGTGCTCTTCCGATCTCTG 571ATCGCCAC CCAGCTCCAGAAGATGT IL22 TGGGAAGCCAAA 572CAGACGTGTGCTCTTCCGATCTGGA 573 CTCCATCAT AACCAATGCCACTTTTGT IL17AGCCTTCAAGACT 574 CAGACGTGTGCTCTTCCGATCTGCC 575 GAACACCGACCTCAGAGATCAACAGAC IL17F TTGGAGAAGGTG 576 CAGACGTGTGCTCTTCCGATCTCTT 577CTGGTGAC ACCCAGTGCTCTGCAAC TGFB1 TATTCCTTTGCC 578CAGACGTGTGCTCTTCCGATCTACC 579 CGGCATCA TTGGGCACTGTTGAAGT CCL20ACTTGCACATCA 580 CAGACGTGTGCTCTTCCGATCTTCC 581 TGGAGGGTATAAGCTATTTTGGTTTAGTGC IL12A GGTCCCTCCAAA 582 CAGACGTGTGCTCTTCCGATCTGAA583 CCGTTGTC CTAGGGAGGGGGAAAGAAG CXCL12 TGGGAGTTGATC 584CAGACGTGTGCTCTTCCGATCTCTC 585 GCCTTTCC ATTCTGAAGGAGCCCCAT CCL3TGGACTGGTTGT 586 CAGACGTGTGCTCTTCCGATCTCTC 587 TGCCAAACTGAGAGTTCCCCTGTCC CCL14 TTCCTCCTCATC 588 CAGACGTGTGCTCTTCCGATCTCTT 589ACCATCGC ACCACCCCTCAGAGTGC CCL18 GAAGCTGAATGC 590CAGACGTGTGCTCTTCCGATCTGTC 591 CTGAGGGG CCATCTGCTATGCCCA CCL17GAGTGCTGCCTG 592 CAGACGTGTGCTCTTCCGATCTCTC 593 GAGTACTTACCCCAGACTCCTGACT IL12B GCTATGGTGAGC 594 CAGACGTGTGCTCTTCCGATCTTCC 595CGTGATTG TCACCCCCACCTCTCTA CXCR3 GACCTCAGAGGC 596CAGACGTGTGCTCTTCCGATCTCCA 597 CTCCTACT ATATCCTCGCTCCCGG IL33RTTCAGGACTCCC 598 CAGACGTGTGCTCTTCCGATCTAGG 599 TCCAGCATTACCAAATGCCTGTGCC IL4R TGAACTTCAGGG 600 CAGACGTGTGCTCTTCCGATCTTCC 601AGGGTGGT TCGTATGCATGGAACCC CCR4 CCAAAGGGAAGA 602CAGACGTGTGCTCTTCCGATCTATT 603 GTGCAGGG CTGTATAACACTCATATCTTTGCC IL23RAGAATCATTAGG 604 CAGACGTGTGCTCTTCCGATCTCTG 605 CCAGGCGTGGCCAATATGCTGAAACCC IL21R ATTTGAGGCTGC 606 CAGACGTGTGCTCTTCCGATCTAGA 607AGTGAGCT CAAGAGCTGGCTCACCT CXCR5 CCTCCCCAGCCT 608CAGACGTGTGCTCTTCCGATCTTCC 609 TTGATCAG TCGCAAGCTGGGTAATC IL6RCCAGCACCAGGG 610 CAGACGTGTGCTCTTCCGATCTACA 611 AGTTTCTAGCATGTCACAAGGCTGT CXCL13 AGGCAGATGGAA 612 CAGACGTGTGCTCTTCCGATCTGCA 613CTTGAGCC TTCGAAGATCCCCAGACTT LIF TCCCCATCGTCC 614CAGACGTGTGCTCTTCCGATCTTTG 615 TCCTTGTC CCGGCTCTCCAGAGTA PTPRCv1GTTCCCATGTTA 616 CAGACGTGTGCTCTTCCGATCTTAC 617 (CD45RA) AATCCCATTCATCAGGAATGGATGTCGCTAATCA PTPRCv2 ACCCTCTCTCCC 618CAGACGTGTGCTCTTCCGATCTTAG 619 (CD45RO) TCCCTTTC TTGGCTATGCTGGCATG IL10CCCCAACCACTT 620 CAGACGTGTGCTCTTCCGATCTTTC 621 CATTCTTGAATTCCTCTGGGAATGTT CD40LG CCTCCCCCAGTC 622 CAGACGTGTGCTCTTCCGATCTGAG 623TCTCTTCT TCAGGCCGTTGCTAGTC

In the unstimulated sample, PCA analysis revealed two major subsets ofcells. A closer look at the genes enriched in each subset showed thatone subset represented CD8+ cells with expression of CD8A, CD8B, NKG2D,GZMA, GZMH, GZMK, and EOMES, and the other subset represented CD4+ cellswith expression of CD4, CCR7 and SELL (FIG. 44A and FIG. 45).

In the stimulated sample, two branches of cells were immediately clearon the PCA plot (FIG. 44B and FIG. 46A-D). The first principal componentrepresented the degree of response of individual cells to stimulant interms of varying level of expression of IFNG, TNF, CD69, and GAPDH.Expression of CCL3, CCL4, and GZMB, which are cytokines and effectormolecules associated with cytotoxic T cells, and LAG3, a markerassociated with exhausted cells, was localized to cells in the upperbranch. Expression of IL2, LTA, CD40LG, and CCL20, which are cytokinesassociated with helper T cells, was localized to the lower branch. Othergenes that have been known to be upregulated in activated T cells,including ZBED2, IL4R, PRDM1, TBX21, MYC, FOSL1, CSF2, TNFRSF9, BCL2 andFASLG, were expressed in various degrees in a smaller number of cells(FIG. 46A-D). Most of these cytokines, effector molecules, andtranscription factors were not expressed or were expressed at very lowlevels by cells in the unstimulated sample. While most of the cells thatresponded within this short period of stimulation were presumably memorycells, we observed a small population of cells that produced lower levelof IL2 and not other cytokines nor effector molecules, and may representnaive cells (FIG. 44B, arrow).

To fully appreciate the heterogeneity in response, we clustered thecells based on a pair-wise correlation coefficient. While the two maingroups of CD4 and CD8 cells were obvious, there was considerablediversity within each set in terms of the combination and level ofactivated genes expressed (FIG. 47 and FIG. 48).

We observed that there were a few cytokines, namely IL4, IL5, IL13,IL17F, IL22, LIF, IL3, and IL21, that were upregulated by a few hundredor more folds in the stimulated sample as a whole as compared to theunstimulated one, but were contributed only by a few cells in the sample(FIG. 44C). Subsets of these cytokines were expressed by the same cells(FIG. 49A-C). For instance, the same single cell contributed to most ofthe counts of IL17F and IL22, which were signatures for Th17 cells.Another 7 cells expressed various combinations of IL4, IL5, IL13, whichwere signatures of Th2 cells, and expressed various combinations ofthem. Such observation highlights the importance of large-scale singlecell analysis, especially when the contribution to overall expressionchanges was derived from a rare subpopulation.

We repeated the same stimulation experiment with T cells from a secondblood donor and analyzed the profile of 669 and 595 single cells in thestimulated and unstimulated sample, respectively. While the overalllevel of activation was lower (smaller magnitude in terms of change inexpression) in this individual (possibly indicating inter-individualvariability to stimulation), we observed the same trends in PCAanalysis, as well as heterogeneity in individual cell's response tostimulus (FIG. 48).

Identification of Rare Antigen Specific T Cells

We demonstrated the utility of our platform to identify rare cells usingthe model of antigen specific cells in CD8+ T cell population. Weexposed fresh blood of the same two blood donors who were seropositivefor cytomegalovirus (CMV) to CMV pp65 peptide pool. A separate untreatedblood aliquot of each donor served as negative control. We subsequentlyisolated CD8+ T cells and analyzed the response of stimulated andunstimulated cells on our platform. We obtained data from 2274, 2337,581, and 253 cells in donor 2's CMV stimulated and unstimulated, anddonor 1's CMV stimulated and unstimulated samples, respectively.

Except for donor 1's negative control that yielded relatively smallnumber of cells to form obvious clusters in clustering analysis, all therest of the samples showed two main groups of cells (FIGS. 50A, 51 and52). Cells in one group expressed naive cell and central memoryassociated markers SELL, CCR7, and CD27, while cells in the other groupexpressed effector memory cell (CCL4, CX3CR1, CXCR3) and effector cellassociated genes (EOMES, GZMA, GZMB, GZMH, TBX21, ZNF683). There was adistinct small subset of cells that occupy space in between the twobranches and express granzyme K (GZMK), as well as another subset ofHLA-DRA expressing cells. The differential expression of the differenttypes of granzymes has previously been reported (8). Our resultsrecapitulated those observed in previous CyTOF experiments with CD8+ Tcells (9).

While a considerable proportion of cells seemed to respond to theexposure to the antigen via expression CD69 and MYC (FIG. 52), we foundonly a few cells that expressed IFNG, a signature cytokine for activatedantigen specific cell. Most of the IFNG expressing cells were also amongthose cells carried the most total detected transcript molecules in thegene panel, an indication of active cell state, and belong to theeffector memory/effector cell cluster (FIGS. 50B and 53). We identified5 out of 581 (0.86%) and 2 out of 2274 (0.09%) cells in donors 1 and 2respectively that were likely to be CMV specific based on IFNGexpression and overall transcription level. Among those cells, there wassubstantial amount of heterogeneity in terms of combinations and levelsof effector molecules (e.g., granzymes) and cytokines (e.g., IFNG, IL2,CCL3, CCL4, TNF, CSF2, IL4) expressed (FIG. 54). Interesting, the singlecell that expressed most transcripts in donor 2 expressed both IL6 andIL1B but not IFNG.

Discussion

In this example, we presented highly scalable mRNA cytometry that used arecursive Poisson strategy to isolate single cells, to uniquely barcodecellular content, and to barcode individual molecules for quantitativeanalysis. We have shown that we may simultaneously identify and counttranscript molecules belonging to each cell in a sample containing a fewthousands cells. Further, we have demonstrated to use of this techniqueto characterize individual cells based on their expression profiles innaturally occurring heterogeneous systems, and detection of rare cellsin a large background population.

The throughput and simplicity of CytoSeq presents a major advance overexisting approaches involving microtiter plates or microfluidic chipsfor sequencing based measurement of gene expression of single cells.Because the experimental procedure is simple and reagent consumption percell is low (in the nanoliter range), it enables one to readily carryout single cell analysis for large number of cells across multipleconditions. In this study alone, we performed gene expression profilingof a total of .about.14,600 single cell across 12 experiments, whichwould be costly and time-consuming if carried out by existingapproaches. The number of cells measured by CytoSeq may be furtherscaled up simply by increasing the size of the microwell array and thelibrary size of the barcoded beads, which is readily achieved bycombinatorial synthesis. In addition, there is no restriction on theuniformity of cell sizes, thus allowing direct analysis of complexsamples containing cells with a variety of cell sizes and shapes, suchas PBMCs shown in this example, without any pre-sorting.

CytoSeq data resembled those of flow cyometry (FC), but with importantdifferences. First, CytoSeq offers more versatility in terms of thenumber and type of gene products studied. Unlike flow cytometry that isconfined mostly to a handful of surface proteins and requires optimallybinding antibodies, CytoSeq allowed measurement of any transcribed mRNAsvia nucleic acid amplification techniques. Optimal primer design andassay conditions enable us to routinely achieve .about.88% mapped ratevia multiplex PCR for an arbitrarily chosen panel of 100 or more genes(Table 21). Additionally, the entire transcriptome of each single cellin the sample may also be measured via universal amplification of thebead bound cDNA, although one has to be mindful with the relatively lowefficiency of commonly used universal amplification techniques (7) andthe high sequencing depth required for measuring the whole transcriptomeacross thousands of cells.

Second, in contrast to flow cytometry that relies on the kinetics ofantibody binding, CytoSeq provides digital, absolute readout of geneexpression level through molecular indexing. It has higher sensitivityand specificity to a single rare cell event because the detection wasachieved by the co-expression of large number of genes specific to therare cells. It therefore consumes much smaller amount of sample ascompared to flow cytometry that requires certain number of events inorder to form reliable clusters for gating.

Our data illustrates the importance of single cell versus bulk analysis.For instance, we showed scenarios where the most highly expressed genesin a sample of thousands of cells as whole were contributed by only oneor a few cells. Most importantly, our experiments illustrate theimportance of examining both large number of cells and large number ofgenes in single cell gene expression studies, an ability that isextremely limiting in prior approaches. The availability of such a toolfor the routine measurement of expression across thousands of singlecells in a biological sample may help accelerate the understanding ofcomplex biological systems and drive novel applications in clinicaldiagnostics, such as circulating tumor cell analysis and immuneresponses monitoring. We envision that our massive parallel single cellbarcoding regime may also be adopted to measure the genome, as well asthe genome and the transcriptome simultaneously, for studying singlecell genome instability in areas such as cancer biology andneuroscience.

TABLE 21 number of reads with number of number of exact match uniquecell number of reads with % reads to a cell % read after barcodes thatreads exactly 1 aligned to barcode and gene and satisfy associated totalnumber match to one gene in alignment to barcode filtering with thoseExperiment of reads gene in panel the panel one gene alignment criteriacell barcodes K562 + Ramos 2399025 2154454 90% 1175715 49% 768 859470Primary B + Ramos 5711013 5203308 91% 3495392 61% 1198 2868577 PBMC1270214 1105687 87% 803151 63% 632 670576 PBMC replicate 3927672 346853888% 2459367 63% 731 1920956 Donor 1 3529898 3249998 92% 2122416 60% 35171466000 antiCD3/antiCD28 stimulated Donor 1 1557996 1292211 83% 93909460% 1478 719351 antiCD3/antiCD28 negative control Donor 2 606865 55287791% 403943 67% 669 246234 antiCD3/antiCD28 stimulated Donor 2 332951283723 85% 205762 62% 595 86866 antiCD3/antiCD28 negative control Donor1 CMV 1064648 958410 90% 697057 65% 581 401629 stimulated Donor 1 CMV619957 547259 88% 406801 66% 253 192605 negative control Donor 2 CMV1902977 1692734 89% 1229667 65% 2274 688296 stimulated Donor 2 CMV1671419 1346637 81% 977344 58% 2337 715453 negative controlSynthesis of Bead Library

Beads were manufactured by Cellular Research, Inc. using a split-poolcombinatorial approach. Briefly, twenty-micron magnetic beadsfunctionalized with carboxyl groups were distributed into a 96 tubescontaining oligos with 5′ amine, followed by a universal sequence, firstpart of the cell label that is different for different tubes, and alinker sequence. The oligos were covalently coupled onto the beads bycarbodiimide chemistry. Beads were pooled and split into a second set of96 tubes containing oligos with a second linker sequence on the 5′ end,followed by the second part of the cell label that is different fordifferent tubes, and complementary sequence to the first linker. Oligoson the beads were extended by DNA polymerase upon hybridization tooligos in solution via the first linker. Beads were pooled and splitinto a third set of 96 tubes containing oligos with oligo(dA) on the 5′end, followed by a randomer sequence that serves as the molecular label,the third part of the cell label, and a complementary sequence to thesecond linker. Oligos on the beads were extended by DNA polymerase uponhybridization to oligos in solution via the second linker. The finalbead library has a size of 96×96×96 (884,736) cell labels.

Fabrication of Microwell Array

Microwell arrays were fabricated using standard photolithography. Arraysof pillars were patterned on photoresist on silicon wafer. PDMS waspoured onto the wafer to create arrays of microwells. Replicas of thewafer were made with NOA63 optical adhesive using PDMS microwell arrayas template. Agarose (5%, type IX-A, Sigma) microwell arrays were castedfrom the NOA63 replica before each experiment.

Sample Preparation

K562 and Ramos cells were cultured in RPMI-1640 with 10% FBS and 1antibiotic-antimycotic. Primary B cells from a healthy donor werepurchased from Sanguine Biosciences. PBMCs from a healthy donor wereisolated from fresh whole blood in sodium heparin tube acquired from theStanford Blood Center using Lymphoprep solution (StemCell).

T Cell Stimulation

Heparinized whole blood of two CMV seropositive blood donors wasobtained from the Stanford Blood Center. For CMV stimulation, 1 ml ofwhole blood was stimulated with CMV pp65 peptide pool diluted in PBS(Miltenyi Biotec) at a final concentration of 1.81 μg/ml for 6 hours at37 C. A separate aliquot of whole blood of each donor was incubated withPBS as negative controls. CD8+ T cells were isolated using RosetteSepcocktail (StemCell) and subsequently deposited onto microwell arrays.For anti-CD3/anti-CD28 stimulation, T cells from the same two donorswere isolated from whole blood using RosetteSep T cell enrichmentcocktail and resusupended in RPMI-1640 with 10% FBS and1×antibiotic-antimycotic. One aliquot of cells from each donor wasincubated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies)at .about.1:1 bead to cell ratio at 37 C for 6 hours. A separate aliquotof cells from each donor were placed in incubator with no stimulationand served as negative control.

Single Cell Capture

Single cell suspension was pipetted on to the microwell array at adensity of .about.1 cell per 10 microwells. After washing to removeuncaptured cells, magnetic beads were loaded at a density of .about.5beads per well to saturate the microwell array. After washing to removeexcess beads, cold lysis buffer (0.1M Tris-HCl pH 7.5, 0.5M LiCl, 1%LiSDS, 10 mM EDTA, 5 mM DTT) was pipetted over the surface of themicrowell array. After 10 minutes of incubation on a slide magnet, beadswere retrieved from the microwell array. Beads were collected in amicrocentrifuge tube, and washed twice with wash A buffer (0.1MTris-HCl, 0.5M LiCl, 1 mM EDTA) and once with wash B buffer (20 mMTris-HCl pH 7.5, 50 mM KCl, 3 mM MgCl2). From this point forward, allreactions were carried out in a single tube.

cDNA Synthesis

Washed beads were resupsended in 404 RT mix (1×First Strand buffer, 1 μLSuperRase Inhibitor, 1 μL SuperScript II or SuperScript III, 3 mMadditional MgCl2, 1 mM dNTP, 0.2 ug/μL BSA) in a microcentrifuge tubeplaced on a rotor in a hybridization oven at temperatures 50 C for 50minutes (when using SuperScript III for the early experiment with K562and Ramos cells) or 42 C for 90 minutes (when using Superscript II forthe rest of the experiments). Beads were treated with 1 μL of ExoI (NEB)in 20 μL of 1×ExoI buffer at 37° C. for 30 minutes, and 80° C. for 15minutes.

Multiplex PCR and Sequencing

Each gene panel contained two sets of gene specific primers designed byPrimer3. A custom MATLAB script was written to select PCR primers suchthat there was minimal 3′ end complementarity across the primers withinthe set. Primers in each panel are listed in Table 21. The amplificationscheme is shown in FIG. 55. PCR were performed with the beads with KAPAFast Multiplex Kit, with 50 nM of each gene specific primer in the firstprimer set and 400 nM universal primer, in a volume of 100 μL or 200 μL,with the following cycling protocol: 3 min at 95 C; 15 cycles of 15 s at95 C, 60 s at 60 C, 90 s at 72 C; 5 min at 72 C. Magnetic beads wererecovered and PCR products were purified with 0.7×Ampure XP. Half of thepurified products were used for the next round of nested PCR with thesecond primer set using the same KAPA kit and cycling protocol. Afterclean up with 0.7×Ampure XP, 1/10^(th) of the product was input into afinal PCR reaction whereby the full-length Illumina adaptors wereappended (1×KAPA HiFi Ready Mix, 200 nM of P5, 200 nM of P7. 95 C 5 min;8 cycles of 98 C 15 s, 60 C 30 s, 72 C 30 s; 72 C 5 min).

Data Analysis

Sequencing of library was performed on Illumina MiSeq instrument with150×2 by chemistry at a median depth of 1.6 million reads per sample.Sequencing revealed the cell label, the molecular label, and the gene ofeach read (FIG. 55). The assignment of gene of each read was done withthe alignment software ‘bowtie’ (ref). The cell and molecular labels ofeach read were analyzed using custom MATLAB scripts. Reads were groupedfirst by cell label, then by gene and molecular label. To calculate thenumber of unique molecules per gene per cell, the molecular labels ofreads with the same cell label and gene assignment were clustered. Editdistance greater than 1 base was considered as a unique cluster, andthus a unique transcript molecule. A table containing digital geneexpression information of each cell was constructed for each sample—eachrow in the table represented a unique cell label, each columnrepresented a gene, and each entry in the table represented the count ofunique molecules within a gene per cell label. The table was filteredsuch that unique molecules that were sequenced only once (i.e.redundancy=1) were removed. Subsequently, cells with a sum of uniquemolecules less than 10 or with co-expression of 4 or less genes in thepanel were removed. The filtered table was then used for clusteringanalysis. Principal component analysis and hierarchical clustering wasperformed on log-transformed transcript count (with pseudocount of 1added) with built-in functions in MATLAB.

References cited in Example 15, all of which are incorporated byreference in their entireties:

-   A. K. Shalek et al., Single-cell transcriptomics reveals bimodality    in expression and splicing in immune cells. Nature 498, 236 (Jun.    13, 2013).-   S. C. Bendall et al., Single-cell mass cytometry of differential    immune and drug responses across a human hematopoietic continuum.    Science 332, 687 (May 6, 2011).-   A. R. Wu et al., Quantitative assessment of single-cell    RNA-sequencing methods. Nature methods 11, 41 (January, 2014).-   B. Treutlein et al., Reconstructing lineage hierarchies of the    distal lung epithelium using single-cell RNA-seq. Nature 509, 371    (May 15, 2014).-   S. Islam et al., Characterization of the single-cell transcriptional    landscape by highly multiplex RNA-seq. Genome research 21, 1160    (July, 2011).-   G. K. Fu, J. Hu, P. H. Wang, S. P. Fodor, Counting individual DNA    molecules by the stochastic attachment of diverse labels.    Proceedings of the National Academy of Sciences of the United States    of America 108, 9026 (May 31, 2011).-   G. K. Fu, J. Wilhelmy, D. Stern, H. C. Fan, S. P. Fodor, Digital    encoding of cellular mRNAs enabling precise and absolute gene    expression measurement by single-molecule counting. Analytical    chemistry 86, 2867 (Mar. 18, 2014).-   K. Bratke, M. Kuepper, B. Bade, J. C. Virchow, Jr., W. Luttmann,    Differential expression of human granzymes A, B, and K in natural    killer cells and during CD8+ T cell differentiation in peripheral    blood. European journal of immunology 35, 2608 (September, 2005).-   E. W. Newell, N. Sigal, S. C. Bendall, G. P. Nolan, M. M. Davis,    Cytometry by time-of-flight shows combinatorial cytokine expression    and virus-specific cell niches within a continuum of CD8+ T cell    phenotypes. Immunity 36, 142 (Jan. 27, 2012).

Example 16 Development of Single Cell Quantification Protocol

FIG. 56 depicts a general workflow for the quantification of RNAmolecules in a sample. In this example, the total number of RNAmolecules in the sample was equivalent to the total number of RNAmolecules in a single cell. As shown in Step 1 of FIG. 56, RNA molecules(110) were reverse transcribed to produce cDNA molecules (105) by thestochastic hybridization of a set of molecular identifier labels (115)to the polyA tail region of the RNA molecules. The molecular identifierlabels (115) comprised an oligodT region (120), label region (125), anduniversal PCR region (130). The set of molecular identifier labelscontained 960 different types of label regions.

Part I. Reverse Transcription and Labeling of RNA Molecules

An RNA sample was prepared by mixing the following:

number of RNA Genes molecules Lys (spike-in control) 456 Phe (spike-incontrol) 912 Thr (spike-in control) 1824 Dap (spike-in control) 6840 Kan(spike-in control) 7352 Lymphocyte cell line RNA 10 pg (1 cellequivalent) MS2 carrier (no polyA) 6 × 10¹¹

RNA molecules were labeled by preparing in an eppendorf tube a labelingmix as follows:

Amount (μL) RNA sample 2 ms2 RNA 1 μg/μL 1 10 mM dNTP 1 960 dT oligospool (set#4) 10 μM 0.4 water 9.1

Note: dT oligos pool (set #4) refers to the set of molecular identifierlabels.

The molecular identifier labels were hybridized to the RNA molecules byincubation at 65° C. for 5 minutes. The labeling mix was stored on icefor at least 1 minute.

The labeled RNA molecules were reverse transcribed by the addition ofthe reverse transcription mix as described below:

Amount (μL) 5X first strand buffer 4 0.1M DTT 1 superase-in 20 u/μL 0.5superscript III RT 1

Once the reverse transcription mix was added to the eppendorf tubecontaining labeling mix reaction, the reverse transcription reaction wasconducted by incubating the sample at 37° C. for 5 minutes, followed byincubation at 50° C. for 30 minutes, and lastly incubation at 75° C. for15 minutes. Reverse transcription of the labeled RNA molecules producedlabeled cDNA molecules (170).

Once the RNA molecules were reverse transcribed and labeled, excessoligos were removed from the sample by Ampure bead purification (Step 2of FIG. 1). Ampure bead purification was performed by adding 20 μl ofampure beads to the eppendorf tube containing the reverse transcribedand labeled RNA molecules and incubating the tube at room temperaturefor 5 minutes, The beads were washed twice with 70% ethanol to removethe excess oligos. Once the excess oligos were removed by the ethanolwashes, 20 μl of 10 mM Tris was added to the tube containing thebead-bound labeled cDNA molecules.

As shown in Step 3 of FIG. 56, the labeled cDNA molecules (170) wereamplified by multiplex PCR. Custom amplification of the labeled cDNAmolecules was performed by using a custom forward primer (F1, 135 inFIG. 1) and a universal PCR primer (140). Table 23 lists the 96different custom forward primers that were used to amplify 96 differentgenes to produce labeled amplicons (180) in a single reaction volume.

In order to optimize multiplex PCR reactions, 3 multiplex PCR reactionsmixtures were prepared. Multiplex PCR reaction 1 was prepared asfollows:

Reaction 1 Amount (μL) 10X titanium 5 10 mM dNTP 1.5 water 35.5 1 μMeach F1 primer pool 5 PCR004 10 μM 1 purified cDNA 1 Titanium polymerase1

The reaction condition for Multiplex PCR reaction was 1 cycle at 94° C.for 2 min, followed by 25 cycles of 94° C. for 30 sec, 57° C. for 60sec, and 68° C. for 1 min, then 1 cycle of 68° C. for 7 min and 1 holdcycle at 4° C.

Multiplex PCR reactions 2 and 3 were prepared as follows:

Reaction 2 Reaction 3 Amount (μL) Amount (μL) 2X Qiagen Multiplex mix 2525 1 μM each F1 primer pool 5 5 PCR004 10 μM 1 1 Q solution 5 water 1813 purified cDNA 1 1

The multiplex PCR reaction condition for Reactions 2 and 3 was 1 cycleat 95° C. for 15 min, followed by 25 cycles of 94° C. for 30 sec, 57° C.for 90 sec, and 72° C. for 1 min, then 1 cycle of 68° C. for 7 min and 1hold cycle at 4° C.

The F1 primer pools contained the following primers:

F1 PCR Primers Sequence SEQ ID NO: 100611KanF2 CTGCCTCGGTGAGTTTTCTC 624Lys_L_269 CTTCCCGTTACGGTTTTGAC 625 phe_L_177 AAAACCGGATTAGGCCATTA 626thr_L_332 TCTCGTCATGACCGAAAAAG 627 dap_L_276 CAACGCCTACAAAAGCCAGT 628

Kan, Phe and Dap control genes were selectively amplified by nested PCR.Nested PCR amplification reactions were prepared as follows:

Multiplex PCR Rxn # 1 2 3 1 2 3 1 2 3 PCR Rxn # 1 2 3 4 5 6 7 8 9 μL μLμL μL μL μL μL μL μL 10x Taq 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 10 mMdNTP 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 water 22.25 22.25 22.25 22.2522.25 22.25 22.25 22.25 22.25 Cy3 PCR004 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.5 10 μM KanF3_5P 5 μM 1 1 1 Phe_L_215 5 μM 1 1 1 Dap_L_290 5 μM 1 1 1Multiplex PCR Rxn 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 USB taq 0.25 0.250.25 0.25 0.25 0.25 0.25 0.25 0.25 Note: The multiplex PCR reaction usedfor PCR reactions 1, 4, and 7 was multiplex PCR reaction # 1. Themultiplex PCR reaction used for PCR reactions 2, 5 and 8 was multiplexPCR reaction # 2. The multiplex PCR reaction used for PCR reactions 3, 6and 9 was multiplex PCR reaction # 3.

The primers used for nested PCR are disclosed as follows:

Nested SEQ PCR primer Sequence ID NO: KanF4_5P/5Phos/GTGGCAAAGCAAAAGTTCAA 629 Phe_L_215 TGAGAAAGCGTTTGATGATGTA 630Dap_L_290 GCCAGTTTATCCCGTCAAAG 631

The PCR amplification reaction condition for Reactions 1-9 was 1 cycleof 94° C. for 2 min, 30 cycles of 94° C. for 20 sec, 55° C. for 20 sec,and 72° C. for 20 sec, then 1 cycle at 72° C. for 4 min and 1 hold cycleat 4° C.

The 4 μl of PCR products of PCR amplification reactions 1-9 were run onan agarose gel. As shown in FIG. 58A, Reactions 1-3 showed the presenceof the Kan control gene, Reactions 4-6 showed the presence of the Phecontrol gene, and Reactions 7-9 showed the presence of the Dap controlgene.

The PCR products from PCR reactions 1-9 were prepared for hybridizationto an Applied Microarray Inc. (AMI) array. Hybridization mixtures wereprepared as follows:

μL PCR product 20 Wash A (6X SSPE + 0.01% Triton X-100) 55 Cy3 Oligo(760 pM) 1

The hybridization mixtures 1-9, corresponding to the mixtures containingPCR products from PCR reactions 1-9, respectively, were denatured at 95°C. for 5 minutes and then placed at 4° C. The hybridization mixtureswere transferred to an AMI array slide and incubated overnight at 37° C.

After the overnight hybridization, the AMI array slide was washed andthen scanned. Theoretical and actual measurements and percent accuracyare depicted below:

Hybridization mixtures # 1 2 3 4 5 6 7 8 9 Multiplex Titanium QiagenQiagen + Titanium Qiagen Qiagen + Titanium Qiagen Qiagen + PCR (1) (2) Q(3) (1) (2) Q (3) (1) (2) Q (3) condition (Rxn #) Theoretical 3676(bioanalyzer) 912 6840 measurement Actual 1826 1740 2116 299 235 2511165 1077 172 measurement % detection 49.7 47.3 57.6 32.8 25.8 27.5 1715.7 2.5 Note: The theoretical measurement is based on detection of 100%of the Kan, Phe and Dap control genes.

PCR products from Reaction 2 were purified by Ampure purification.Ampure purification was performed as follows:

μL F1 PCR products from X01 sample 2 30 Ampure beads 30

Ampure purification reactions were incubated at room temperature for 5min and then washed in 70% ethanol. Purified PCR products were elutedfrom the beads in 30 μl of water. The concentration of the PCR productswas 6 ng/μL as determined by a Nanodrop spectrometer.

Part II: Library Preparation Protocol

PCR products purified from the X01 sample 2 (see Example 1) were used toprepare a DNA library. An F2 primer pool was created from mixing thefollowing primers:

F2 PCR Primers Sequence SEQ ID NO: Lys_L_269 CTTCCCGTTACGGTTTTGAC 632phe_L_177 AAAACCGGATTAGGCCATTA 633 thr_L_332 TCTCGTCATGACCGAAAAAG 634dap_L_276 CAACGCCTACAAAAGCCAGT 635

An F2 primer mix was prepared by mixing the following

F2 primer mix μL water 750 F2 primer pool 1 uM each/100 uM total 100

The F2 primer mix was incubated at 95° C. for 3 min and then stored onice. The following ligation mix was added to the F2 primer mix toproduce an F2 primer ligation mix:

Ligation mix μL 10X DNA ligase buffer NEB 100 T4 PNK USB 50

The F2 primer ligation mix was incubated at 37° C. for 1 hour, followedby an incubation at 65° C. for 20 min. The F2 PCR primers were ethanolprecipitated and the concentration of the primer pool was determined bya Nanodrop spectrophotometer. The F2 primer pool was resuspended toproduce a final concentration of 1 uM each/100 uM total.

As shown in Step 4 of FIG. 56, the labeled amplicons (180) wereamplified by multiplex PCR. 96 different custom forward primers (F2, 145in FIG. 1) and a universal PCR primer (140) were used to amplify thelabeled amplicons (X01 sample 2 from Example 1) in a single reactionvolume. Table 24 lists the 96 different custom forward primers.

The multiplex PCR reaction was prepared as follows:

Multiplex PCR mix μL 2X Qiagen Multiplex mix 25 1 μM each F2 primer poolkinase 5 PCR004 5′P 10 μM 1 water 18 purified first PCR X01 sample 2 1

The multiplex PCR condition was 1 cycle at 95° C. for 15 min, followedby 18 cycles of 94° C. for 30 sec, 57° C. for 90 sec, and 72° C. for 1min, then 1 cycle of 68° C. for 7 min and 1 hold cycle at 4° C. Themultiplexed amplicons were purified by Ampure purification and elutedwith 50 μL of water. The concentration of the amplicons was determinedto be 30 ng/μL by a Nanodrop spectrophotometer. 5 μL of the ampliconswas run on an agarose gel (FIG. 58B).

As shown in Step 5 of FIG. 56, adaptors (150, 155) were ligated to thelabeled amplicons (180) to produce adaptor labeled amplicons (190).Adaptor labeled amplicons were produced as follows:

Adaptor mix μL 10X T4 ligase USB 10 water 60 purified nested PCR product10 annealed, pooled 96 ABC adaptors 50 μM 10 T4 DNA ligase (3 μl neb hc,7 μl usb) 10

The adaptor mix was incubated at 16° C. for 4 hours. The adaptor labeledamplicons were purified by Ampure purification and eluted in 20 μL of 10mM Tris.

The purified adaptor labeled amplicons were gap-repaired and PCRamplified as follows:

Fill-in and PCR mix μL 10x thermoPol buffer 5 10 mM dNTP 1.5 water 32 CRP1 10 μM 3 CR DX D1 10 μM 3 purified adaptor labeled amplicons 5 Ventexo-2 u/μL 0.5

The PCR condition was 1 cycle of 72° C. for 2 min, followed by 94° C.for 1 min, 12 cycles of 94° C. for 15 sec, 60° C. for 15 sec and 72° C.for 30 sec, 1 cycle of 72° C. for 4 min and 1 hold cycle at 4° C. ThePCR products were purified by Ampure purification and eluted in 30 μl ofTE. The concentration of the purified PCR product was 22 ng/μL (83 nM)as determined by Nanodrop spectroscopy. 5 μL of the PCR purifiedproducts were run on a 1% agarose gel (FIG. 58B)

Part III. Sequencing of the Adaptor Labeled Amplicon Library

The adaptor labeled amplicon library was sequenced using a MiSeqSequencer.

A sequence mapping summary is shown below:

Require Perfect Allow 1 bp Match mismatch Total Read Pairs 7,724,955 #of RNA with universal 2,499,444 4,716,378 primer and polyA match (32%)(61%) # of RNA mapped to targets 2,373,700 4,489,485 (96 genes)

As shown in the sequence mapping summary above, many reads were lost dueto the stringent polyA matching criteria. FIG. 59 shows the reads andcounts across all detected genes.

Sequencing reads were also used to quantify specific genes. FIG. 61-62depict a plot of the reads observed per label detected (RPLD) forvarious genes. Conventional rpkm values are also shown in the plotsdepicted in FIG. 61-62. FIG. 59 summarizes a comparison of RPLD and RPKMfor various genes.

FIG. 63 depicts a plot of total reads (labels) versus rpld for variousgenes.

The data represented in FIGS. 4,7 and 8 are also shown in numerical formin Table 25.

FIG. 64 depicts a plot of RPKM for undetected genes.

The quantity of the spike-in controls in the adaptor labeled ampliconlibrary was determined by MiSeq sequencing. Results from MiSeqsequencing of the spike-in controls are shown in the table below.

Spike-in input Labels Control N (mfg) Reads (K) Dap 6840 1,920,503 893Phe 912 470,738 859 Thr 1824 410,664 847 Lys 456 282,174 847 Kan 7352 2423

In the table above, input N refers to the original number of thespike-in control; Reads refers to the total number of read pairs; andLabels (K) refers to the number of different labels detected bysequencing. FIG. 60A-D depicts a plot of the reads observed per labeldetected (RPLD) for Lys, Phe, Thr, and Dap spike-in controls,respectively. FIG. 60E depicts a plot of Reads versus Input.

TABLE 23 SEQ Name Sequence ID NO: NM_144646.3F1 TTGACTTTGCCTTGGAGAGC 636NR_015342.1F1 TTTTTCTTACAGTGTCTTGGCATA 637 NM_000193.2F1CGTGACCCTAAGCGAGGAG 638 NM_001777.3F1 TTTGCAGTGATTTGAAGACCA 639NM_000600.3F1 GGCATTCCTTCTTCTGGTCA 640 NM_021127.2F1CTGGGCTATATACAGTCCTCAAA 641 NM_004318.3F1 GGGGTGATTATGACCAGTTGA 642NM_002467.4F1 TGCATGATCAAATGCAACCT 643 NM_001773.2F1TCTTCCGAAAAATCCTCTTCC 644 NM_001770.5F1 CTGGGGTCCCAGTCCTATG 645NM_001718.4F1 TGTACTGGGAAGGCAATTTCA 646 NR_023920.1F1 GAGCCGCTGGGGTTACTC647 NM_000267.3F1 CAGTTAGTTGCTGCACATGGA 648 NM_000633.2F1TTGCATTTCTTTTGGGGAAG 649 NM_000314.4F1 GTCATGCATGCAGATGGAAG 650NM_021151.3F1 GCTGCAGTGAGCTGTGATGT 651 NM_002415.1F1 GTTCCTCTCCGAGCTCACC652 NM_004985.3F1 TCCGAAAGTTTCCAATTCCA 653 NM_005375.2F1TTGTTTGGGAGACTCTGCATT 654 NM_000555.3F1 GACCCCACTTGGACTGGTAG 655NM_001668.3F1 GTGATCTTGATTGCGGCTTT 656 NM_025237.2F1GGGGGAAAAACTACAAGTGC 657 NM_021117.3F1 TGATTCCTTTTCCTGCCTGT 658NM_016316.2F1 AAAAACCTCCAGGCCAGACT 659 NM_021975.3F1AATCAAAATAACGCCCCAGA 660 NM_004333.4F1 TTGCTAAAAATTGGCAGAGC 661NM_001621.4F1 TTGTTAAGTGCCAAACAAAGGA 662 NM_005239.5F1AAGCTGGGAAGAGCAAAGC 663 NM_000485.2F1 AGGACAGAGGGTGGTCGTC 664NM_004048.2F1 TGAGTGCTGTCTCCATGTTTG 665 NM_001657.2F1CCTCACAGCTGTTGCTGTTATT 666 NM_012238.4F1 AAAACACCCAGCTAGGACCA 667NM_002055.4F1 AACTGAGGCACGAGCAAAGT 668 NM_002392.4F1GCTTTATGGGTGGATGCTGA 669 NM_001625.3F1 ATAATATCGCCAGCCTCAGC 670NM_002110.3F1 TCCAGAGTGTGCTGGATGAC 671 NM_002943.3F1TGCAAGCCATTTATGGGAAT 672 NM_000059.3F1 TGGAATGAGGTCTCTTAGTACAGTT 673NM_018136.4F1 TCCCAGAAACACCTGTAAGGA 674 NM_003467.2F1TGTCTAGGCAGGACCTGTGG 675 NM_004958.3F1 AGTGATGCTGCGACTCACAC 676NM_006139.3F1 GGCTCAGAAAGTCTCTCTTTCC 677 NM_002693.2F1CTCCCAAACTCAGGCTTTCA 678 NM_001080432.2F1 AAAGCGCTGGGATTACAGG 679NM_005954.2F1 CGTCCAGTTGCTTGGAGAAG 680 NM_024865.2F1AATAACCTTGGCTGCCGTCT 681 NM_001905.2F1 GGGAATTCTCAGTGCCAACT 682NM_002046.4F1 GCATCCTGGGCTACACTGAG 683 NM_002253.2F1TGCTGGGAACAATGACTATAAGA 684 NM_002356.5F1 GCCTAAAACACTTTGGGTGGT 685NM_000189.4F1 GGGTGCCCACAAAATAGAGA 686 NM_000546.5F1GAGACTGGGTCTCGCTTTGT 687 NM_152860.1F1 TGGGGAAGGCTTTCTCTAGG 688NM_016231.4F1 TTCAACTTGAGTGATCTGAGCTG 689 NM_000518.4F1TATGGGCAACCCTAAGGTGA 690 NM_000905.3F1 CGCTGCGACACTACATCAAC 691NM_005038.2F1 TGGAGTCTTGCTCTGTCACC 692 NM_000041.2F1 ACGAGGTGAAGGAGCAGGT693 NM_005957.4F1 CGATGCCTTTGGGTAGAGAG 694 NR_002785.2F1ACTGATCGTCCAAGGACTGG 695 NM_000321.2F1 AAAAAGAAATCTGGTCTTGTTAGAAAA 696NM_152756.3F1 TTGAAAAGTGGTAAGGAATTGTGA 697 NM_000610.3F1CACCAAGAATTGATTTTGTAGCC 698 NR_033314.1F1 AAAAATGGGGGAAAATGGTG 699NM_017460.5F1 CATGGTTGAAACCCCATCTC 700 NR_002196.1F1TTCAAAGCCTCCACGACTCT 701 NM_000591.3F1 GCTGGAACAGGTGCCTAAAG 702NM_000106.5F1 CCCTAAGGGAACGACACTCA 703 NM_138712.3F1 ACCTGCTACAAGCCCTGGA704 NM_004304.4F1 GGATCCCTAAGACCGTGGAG 705 NM_000754.3F1CCACCTCAGAGGCTCCAA 706 NM_000492.3F1 TGCTGTATTTTAAAAGAATGATTATGA 707NM_000444.4F1 GTAGCTGGGACGCTGGTTTA 708 NM_002463.1F1ATTCCCTTCCCCCTACAAGA 709 NM_000552.3F1 CCTGAGTGCAACGACATCAC 710NM_005430.3F1 GGGGGAACCAGCAGAAAT 711 NM_003150.3F1 GACCTAGGGCGAGGGTTC712 NM_000388.3F1 AATTCCTGAAGCCAGATCCA 713 NM_007294.3F1AAAATGTTTATTGTTGTAGCTCTGG 714 NM_005933.3F1 TTTCAAGAGCTCAACAGATGACA 715NM_002343.3F1 GACTGCCCGGACAAGTTTT 716 NM_000376.2F1 GAGAAGGTGCCCCAAAATG717 NM_002462.3F1 AGCCACTGGACTGACGACTT 718 NM_021005.3F1GGAGGACTAGTGAGGGAGGTG 719 NM_012343.3F1 GGCAAGTGATGTGGCAATTA 720NM_001741.2F1 GTTGGAGCACCTGGAAAGAA 721 NM_014417.4F1 ATGCCTGCCTCACCTTCAT722 NM_014009.3F1 ACAGGGGCACTGTCAACAC 723 NM_006908.4F1AAAAATCATGTGTTGCAGCTTT 724 NM_005228.3F1 TGCTTTCACAACATTTGCAG 725NM_013994.2F1 AATGTTTCCTTGTGCCTGCT 726 NM_000639.1F1ATATCCTGAGCCATCGGTGA 727 NM_002701.4F1 TTTTGGTACCCCAGGCTATG 728NM_000268.3F1 ACCCCGTGGCATTACATAAC 729 NM_003140.1F1CTTCCAGGAGGCACAGAAAT 730 NM_000551.3F1 CTAACCTGGGCGACAGAGTG 731

TABLE 24 SEQ Name Sequence ID NO: NM_144646.3F2ATATTTGGACATAACAGACTTGGAA 732 NR_015342.1F2 TGCTGACTTTTAAAATAAGTGATTCG733 NM_000193.2F2 GCGGCAGAGTAGCCCTAAC 734 NM_001777.3F2TGGGCTATTTCTATTGCTGCT 735 NM_000600.3F2 AATGGAAAGTGGCTATGCAG 736NM_021127.2F2 GGTTGTAGTCACTTTAGATGGAAAA 737 NM_004318.3F2TTTGTTTGACTTTGAGCACCA 738 NM_002467.4F2 AATGTTTCTCTGTAAATATTGCCATT 739NM_001773.2F2 CACCCCCATATGGTCATAGC 740 NM_001770.5F2 AGCACCAGGTGATCCTCAG741 NM_001718.4F2 TGTTTTGCTGTAACATTGAAGGA 742 NR_023920.1F2TAATGCCACAGTGGGGATG 743 NM_000267.3F2 GGGCCTAAACTTTGGCAGTT 744NM_000633.2F2 TTTTACCTTCCATGGCTCTTTT 745 NM_000314.4F2GCCTTACTCTGATTCAGCCTCTT 746 NM_021151.3F2 CGTAACAAAATTCATTGTGGTGT 747NM_002415.1F2 AGAACCGCTCCTACAGCAAG 748 NM_004985.3F2GTGCTTTCTTTTGTGGGACA 749 NM_005375.2F2 GGGAGTTCTGCATTTGATCC 750NM_000555.3F2 TGGGTCAGAGGACTTCAAGG 751 NM_001668.3F2AGGGTTCTGATCACATTGCAC 752 NM_025237.2F2 CTGCAGGACTGGTCGTTTTT 753NM_021117.3F2 AGGGCAGGGTAGAGAGGGTA 754 NM_016316.2F2TTCTTCCATGCGGAGAAATC 755 NM_021975.3F2 CATGGCTGAAGGAAACCAGT 756NM_004333.4F2 TTGCCAGCTATCACATGTCC 757 NM_001621.4F2TCTTTTCCTGTACCAGGTTTTTC 758 NM_005239.5F2 TGACTGGGAACATCTTGCTG 759NM_000485.2F2 TGGCACCTGTACCCTTCTTC 760 NM_004048.2F2TTCAATCTCTTGCACTCAAAGC 761 NM_001657.2F2 TGGAGTCACTGCCAAGTCAT 762NM_012238.4F2 TTTGCATGATGTTTGTGTGC 763 NM_002055.4F2GCACCCACTCTGCTTTGACT 764 NM_002392.4F2 ACCATGTAGCCAGCTTTCAA 765NM_001625.3F2 GCAACTGGGCATGAGTACCT 766 NM_002110.3F2 CCACACCCCCTTCCTACTC767 NM_002943.3F2 AGTCTGCTTATTTCCAGCTGTTT 768 NM_000059.3F2TCCTGTTCAAAAGTCAGGATGA 769 NM_018136.4F2 AAATCACAAATCCCCTGCAA 770NM_003467.2F2 CTGAACATTCCAGAGCGTGT 771 NM_004958.3F2 CAGTGGGACCACCCTCACT772 NM_006139.3F2 TCTGTAGATGACCTGGCTTGC 773 NM_002693.2F2TCAGAACCAAGATGCCAACA 774 NM_001080432.2F2 CATGACCCAGCCTATGGTTT 775NM_005954.2F2 ACCTCCTGCAAGAAGAGCTG 776 NM_024865.2F2TTGGGAGGCTTTGCTTATTTT 777 NM_001905.2F2 CTGGGAAACACTCCTTGCAT 778NM_002046.4F2 CAACGAATTTGGCTACAGCA 779 NM_002253.2F2CAAAGGTCATAATGCTTTCAGC 780 NM_002356.5F2 TTTGACGTATCTTTTCATCCAA 781NM_000189.4F2 TGTTGTTGGTTTCCAAAAAGG 782 NM_000546.5F2GCCAACTTTTGCATGTTTTG 783 NM_152860.1F2 CCCAAGCTGATCTGGTGGT 784NM_016231.4F2 TGCTGTGAAAGAAACAAACATTG 785 NM_000518.4F2GCACGTGGATCCTGAGAACT 786 NM_000905.3F2 CCAGCCCAGAGACACTGATT 787NM_005038.2F2 CACGCCCAGCTAATTTTTGT 788 NM_000041.2F2CC TGGTGGAAGACATGCAG 789 NM_005957.4F2 TCACACCTGTAATCCCAGCA 790NR_002785.2F2 CAGAGCTCCGCCTCATTAGT 791 NM_000321.2F2TCCATTTCATCATTGTTTCTGC 792 NM_152756.3F2 TGGTGTTTGTAGGTCACTGAACA 793NM_000610.3F2 AACATGGTCCATTCACCTTTATG 794 NR_033314.1F2AGAGCGAGACTCCGTCTCAA 795 NM_017460.5F2 AGTGAGCTGAGATTGCACCA 796NR_002196.1F2 AGACGGCCTTGAGTCTCAGT 797 NM_000591.3F2 GGGAATCCCTTCCTGGTC798 NM_000106.5F2 CTTCCTGCCTTTCTCAGCAG 799 NM_138712.3F2TGCAGGTGATCAAGAAGACG 800 NM_004304.4F2 GGTTTTGAGCATGGGTTCAT 801NM_000754.3F2 CCAGCCCACTCCTATGGAT 802 NM_000492.3F2 AAACTGGGACAGGGGAGAAC803 NM_000444.4F2 TTTGGGTAGGTGACCTGCTT 804 NM_002463.1F2TCACTGAACGAATGAGTGCTG 805 NM_000552.3F2 ACGATGTGCAGGACCAGTG 806NM_005430.3F2 AATTTGCACTGAAACGTGGA 807 NM_003150.3F2CTGTTGTGGCCCATTAAAGAA 808 NM_000388.3F2 TTCCCTCCAGCAGTGGTATT 809NM_007294.3F2 CACCAGGAAGGAAGCTGTTG 810 NM_005933.3F2TTTCCTTGTGTTCTTCCAAGC 811 NM_002343.3F2 TCGCAGGCATTACTAATCTGAA 812NM_000376.2F2 CTCTGGCTGGCTAACTGGAA 813 NM_002462.3F2 AGAGCCCCACCCTCAGAT814 NM_021005.3F2 TGTGCAGAGTTCTCCATCTGA 815 NM_012343.3F2TGCCTGTTACAAATATCAAGGAA 816 NM_001741.2F2 TTTCCCTTCTTGCATCCTTC 817NM_014417.4F2 TGTGACCACTGGCATTCATT 818 NM_014009.3F2CTCACACACACGGCCTGTTA 819 NM_006908.4F2 CACTTGACCAATACTGACCCTCT 820NM_005228.3F2 GTGTGTGCCCTGTAACCTGA 821 NM_013994.2F2CCACTTCCCACTTGCAGTCT 822 NM_000639.1F2 TGTGTGTGTGTGTGTGTGTGT 823NM_002701.4F2 TCTCCCATGCATTCAAACTG 824 NM_000268.3F2TCTAAGTGTTCCTCACTGACAGG 825 NM_003140.1F2 TACTCTGCAGCGAAGTGCAA 826NM_000551.3F2 CCAAGATCACACCATTGCAC 827 NM_144646.3F2ATATTTGGACATAACAGACTTGGAA 828

TABLE 25 Number Number of reads per kb/ Gene of reads labels million(RPKM) APOE 1585 408 0.2 APRT 11280 56 103.4 AREG 147 102 0.0 ASPM 468353 4.4 B2M 209362 698 3891.7 BBC3 8 1 0.0 BCL2 3627 27 33.2 BDNF 12778116 0.3 CD19 38 5 43.1 CD44 6789 47 8.1 COMT 2828 16 10.7 CTPS1 3998 1525.4 CXCR4 10547 54 19.2 CYP3A4 80982 267 0.1 DCX 28 24 0.0 ETS2 6 5 0.0FASLG 3182 565 0.2 FTO 8877 58 11.3 GAPDH 227129 661 3870.8 HCK 294 22.4 HK2 593 2 12.7 IGJ 119449 454 438.9 KDR 2 2 0.0 KRAS 64 31 6.8 LTF126 90 0.0 MARCKS 1563 12 36.9 MIF 17775 89 760.4 MLL 72 9 2.6 MTHFR4854 282 3.9 MX1 100701 285 119.0 MX2 2145 13 45.2 MYB 18361 100 2.8 MYC6859 27 130.5 NF1 4 1 3.7 NNT 15673 78 14.1 PMAIP1 50604 244 126.9 POLG5163 46 7.4 POU5F1 1924 12 1.0 PPID 27354 303 39.0 PTEN 20884 109 51.6RAC1 12454 67 44.8 RB1 1420 14 46.1 RELA 3893 26 17.9 RICTOR 898 5 5.2RORA 954 7 0.1 SOST 1 1 0.0 SP7 1 1 0.0 STAT3 706 28 16.9 TP53 900 3414.6 VHL 11576 106 0.0

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Embodiments

Disclosed herein are methods for analyzing molecules in two or moresamples. The method may comprise: a) producing a plurality ofsample-tagged nucleic acids by: i) contacting a first sample comprisinga plurality of nucleic acids with a plurality of first sample tags toproduce a plurality of first sample-tagged nucleic acids; and ii)contacting a second sample comprising a plurality of nucleic acids witha plurality of second sample tags to produce a plurality of secondsample-tagged nucleic acids, wherein the plurality of second sample tagsare different from the first sample tags; b) contacting the plurality ofsample-tagged nucleic acids with a plurality of molecular identifierlabels to produce a plurality of labeled nucleic acids; and c) detectingat least one of the labeled nucleic acids, thereby determining a countof a plurality of nucleic acids in a plurality of samples. One or moreof the plurality of samples may comprise a single cell or cell lysate.One or more of the plurality of samples may consist of a single cell.The sample tag may comprise a cellular label that identifies the cellfrom which the labeled nucleic acids originated from. The plurality ofsamples consisting of a single cell may be from one or more sources. Thesample tag may comprise a sample index region that identifies the sourceof the single cell. The molecular identifier labels may be referred toas a molecular label. One or more of the plurality of samples maycomprise fewer than 1,000,000 cells. One or more of the plurality ofsamples may comprise fewer than 100,000 cells. One or more of theplurality of samples may comprise fewer than 10,000 cells. One or moreof the plurality of samples may comprise fewer than 1,000 cells. One ormore of the plurality of the samples may comprise fewer than 100 cells.One or more of the plurality of samples may comprise a cell lysate.

Alternatively, the method for analyzing molecules in a plurality ofsamples may comprise: a) producing a plurality of labeled nucleic acidscomprising: i) contacting a first sample with a first plurality ofsample tags, wherein the first plurality of sample tags compriseidentical nucleic acid sequences; ii) contacting the first sample with afirst plurality of molecular identifier labels comprising differentnucleic acid sequences, thereby producing a plurality of first-labelednucleic acids; iii) contacting a second sample with a second pluralityof sample tags, wherein the second plurality of sample tags compriseidentical nucleic acid sequences; iv) contacting the second sample witha second plurality of molecular identifier labels comprising differentnucleic acid sequences, thereby producing a plurality of second-labelednucleic acids, wherein the plurality of labeled nucleic acids comprisesthe plurality of first-labeled nucleic acids and the second-labelednucleic acids; and b) determining a number of different labeled nucleicacids, thereby determining a count of a plurality of nucleic acids in aplurality of samples. The sample tag may comprise a cellular label thatidentifies the cell from which the labeled nucleic acids originatedfrom. The sample tag may comprise a sample index region that identifiesthe source of the single cell. The molecular identifier labels may bereferred to as a molecular label.

Alternatively, the method for analyzing molecules in a plurality ofsamples may comprise: a) contacting a plurality of samples comprisingtwo or more different nucleic acids with a plurality of sample tags anda plurality of molecular identifier labels to produce a plurality oflabeled nucleic acids, wherein: i) the plurality of labeled nucleicacids comprise two or more nucleic acids attached to two or more sampletags and two or more molecular identifier labels; ii) the sample tagsattached to nucleic acids from a first sample of the plurality ofsamples are different from the sample tags attached to nucleic acidmolecules from a second sample of the plurality of samples; and iii) twoor more identical nucleic acids in the same sample are attached to twoor more different molecular identifier labels; and b) detecting at leasta portion of the labeled nucleic acids, thereby determining a count oftwo or more different nucleic acids in the plurality of samples. Thesample tag may comprise a cellular label that identifies the cell fromwhich the labeled nucleic acids originated from. The sample tag maycomprise a sample index region that identifies the source of the singlecell. The molecular identifier labels may be referred to as a molecularlabel.

Further disclosed herein are methods for analyzing molecules in aplurality of samples comprising: a) contacting a first plurality ofmolecules from a first sample of a plurality of samples with a first setof molecular barcodes to produce a first plurality of labeled molecules,wherein a molecular barcode of the first plurality of molecular barcodescomprises a label region and a sample index region; b) contacting asecond plurality of molecules from a second sample of the plurality ofsamples with a second set of molecular barcodes to produce a secondplurality of labeled molecules, wherein a molecular barcodes of thesecond plurality of molecular barcodes comprises a label region and asample index region, and wherein the first plurality of molecularbarcodes and the second plurality of molecular barcodes differ at leastby the sample index region of the molecular barcodes; and c) detectingat least a portion of two or more molecules of the first plurality oflabeled molecules and at least a portion of two or more molecules of thesecond plurality of labeled molecules, thereby determining a count ofthe two or more molecules in the plurality of samples. The firstplurality of molecules may comprise nucleic acid molecules. The secondplurality of molecules may comprise nucleic acid molecules. The labelregion may be referred to as a molecular label. The molecular barcodemay further comprise a cellular label. In instances in which a sample ofthe plurality of samples consists of a single cell, the sample indexregion may refer to the cellular label.

Disclosed herein is a method of selecting a custom primer, comprising:a) a first pass, wherein primers chosen comprise: i) no more than threesequential guanines, no more than three sequential cytosines, no morethan four sequential adenines, and no more than four sequentialthymines; ii) at least 3, 4, 5, or 6 nucleotides that are guanines orcytosines; and iii) a sequence that does not easily form a hairpinstructure; b) a second pass, comprising: i) a first round of choosing aplurality of sequences that have high coverage of all transcripts; andii) one or more subsequent rounds, selecting a sequence that has thehighest coverage of remaining transcripts and a complementary score withother chosen sequences of no more than 4; and c) adding sequences to apicked set until a coverage saturates or a total number of customerprimers is less than or equal to about 96.

Further disclosed herein is a method for producing a labeled moleculelibrary comprising: a) producing a plurality of sample-tagged nucleicacids by: i) contacting a first sample comprising a plurality of nucleicacids with a plurality of first sample tags to produce a plurality offirst sample-tagged nucleic acids; and ii) contacting a second samplecomprising a plurality of nucleic acids with a plurality of secondsample tags to produce a plurality of second sample-tagged nucleicacids, wherein the plurality of first sample tags are different from thesecond sample tags; and b) contacting the plurality of sample-taggednucleic acids with a plurality of molecular identifier labels to producea plurality of labeled nucleic acids, thereby producing a labelednucleic acid library.

Disclosed herein are kits for use in analyzing molecules in a pluralityof samples. The kit may comprise: a) two or more sets of molecularbarcodes, wherein a molecular barcode of the set of one or moremolecular barcodes comprise a sample index region and a label region,wherein (i) the sample index regions of the molecular barcodes of a setof molecular barcodes is the same; and (ii) the sample index regions ofa first set of molecular barcodes are different from the sample indexregions of a second set of molecular barcodes; and b) a plurality ofbeads. The two or more sets of molecular barcodes may be attached to theplurality of beads. The two or more sets of molecular barcodes may beconjugated to the bead. The label region may be referred to as amolecular label. The molecular barcode may further comprise a cellularlabel. In instances in which a sample of the plurality of samplesconsists of a single cell, the sample index region may refer to acellular label.

The kit for analyzing molecules in a plurality of samples may comprise:a) a first container comprising a first plurality of molecular barcodes,wherein: (i) a molecular barcode comprises a sample index region and alabel region; (ii) the sample index regions of at least about 80% of thetotal number of molecular barcodes of the first plurality of molecularbarcodes are identical; and (iii) the label regions of two or morebarcodes of the first plurality of molecular barcodes are different; and(b) a second container comprising a second plurality of molecularbarcodes, wherein: (i) a molecular barcode comprises a sample indexregion and a label region; (ii) the sample index regions of at leastabout 80% of the total number of molecular barcodes of the firstplurality of molecular barcodes are identical; and (iii) the labelregions of two or more barcodes of the first plurality of molecularbarcodes are different; wherein the sample index regions of the firstplurality of molecular barcodes is different from the sample indexregions of the second plurality of molecular barcodes. The label regionmay be referred to as a molecular label. The molecular barcode mayfurther comprise a cellular label. In instances in which a sample of theplurality of samples consists of a single cell, the sample index regionmay refer to a cellular label.

Alternatively, the kit for analyzing molecules in a plurality of samplescomprises: a) a first container comprising a first plurality of sampletags, wherein the plurality of sample tags comprises the same nucleicacid sequence; and b) a second container comprising a first plurality ofmolecular identifier labels, wherein the plurality of molecularidentifier labels comprises two or more different nucleic acidsequences. The label region may be referred to as a molecular label. Ininstances in which a sample of the plurality of samples consists of asingle cell, the sample tag may refer to a cellular label. The kit mayfurther comprise a third container comprising a first plurality ofcellular labels, wherein the plurality of cellular labels comprises twoor more different nucleic acid sequences.

The kits and methods disclosed herein may comprise one or more sets ofmolecular barcodes. The kits and methods disclosed herein may compriseone or more molecular barcodes. The molecular barcodes may comprise asample index region, molecular label region, cellular label region, or acombination thereof. At least two molecular barcodes of a set ofmolecular barcodes may comprise two or more different label regions.Label regions of two or more molecular barcodes of two or more sets ofmolecular barcodes may be identical. Two or more sets of molecularbarcodes may comprise molecular barcodes comprising the same labelregion. In instances in which a sample of the plurality of samplesconsists of a single cell, the sample tag may refer to a cellular label.

The molecular barcodes disclosed herein may comprise a sample indexregion. The sample index region of molecular barcodes of two or moresets of molecular barcodes may be different. The sample index region maycomprise one or more nucleotides. Two or more sequences of sample indexregions of two or more different sets of molecular barcodes may be lessthan about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, or 5% homologous. Two or more sequences ofsample index regions of two or more different sets of molecular barcodesmay be less than about 80% homologous. Two or more sequences of sampleindex regions of two or more different sets of molecular barcodes may beless than about 60% homologous. Two or more sequences of sample indexregions of two or more different sets of molecular barcodes may be lessthan about 40% homologous. Two or more sequences of sample index regionsof two or more different sets of molecular barcodes may be less thanabout 20% homologous.

The molecular barcodes disclosed herein may comprise a cellular label.The cellular label of molecular barcodes of two or more sets ofmolecular barcodes may be different. The cellular label may comprise oneor more nucleotides. Two or more sequences of cellular labels of two ormore different sets of molecular barcodes may be less than about 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% homologous. Two or more sequences of cellular labels oftwo or more different sets of molecular barcodes may be less than about80% homologous. Two or more sequences of cellular labels of two or moredifferent sets of molecular barcodes may be less than about 60%homologous. Two or more sequences of cellular labels of two or moredifferent sets of molecular barcodes may be less than about 40%homologous. Two or more sequences of cellular labels of two or moredifferent sets of molecular barcodes may be less than about 20%homologous.

The molecular barcode disclosed herein may further comprise a universalPCR region. The molecular barcode may further comprise a target-specificregion. The molecular barcode may comprise one or more nucleotides. Thelabel region may comprise one or more nucleotides. The sample indexregion may comprise one or more nucleotides. The universal PCR regionmay comprise one or more nucleotides. The target-specific region maycomprise one or more nucleotides.

The kits and methods disclosed herein may comprise one or more sets ofsample tags. The kits and methods disclosed herein may comprise one ormore sample tags. The sample tags may comprise a sample index region.The sample index region of the sample tags of a first set of sample tagsmay be different from the sample index region of the sample tags of asecond set of sample tags. The sample index region may comprise one ormore nucleotides. Two or more sequences of sample index regions of twoor more different sets of sample tags may be less than about 90%, 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5% homologous. Two or more sequences of sample index regions oftwo or more different sets of sample tags may be less than about 80%homologous. Two or more sequences of sample index regions of two or moredifferent sets of sample tags may be less than about 60% homologous. Twoor more sequences of sample index regions of two or more different setsof sample tags may be less than about 40% homologous. Two or moresequences of sample index regions of two or more different sets ofsample tags may be less than about 20% homologous.

The kits and methods disclosed herein may comprise one or more sets ofmolecular identifier labels. The kits and methods disclosed herein maycomprise one or more molecular identifier labels. The molecularidentifier labels may comprise a label region. The label regions of twoor more molecular identifier labels of a set of molecular identifierlabels may be different. The label region may comprise one or morenucleotides. A sequence of label regions of two or more molecularidentifier labels of a set of molecular identifier labels may be lessthan about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, or 5% homologous. A sequence of label regionsof two or more molecular identifier labels of a set of molecularidentifier labels may be less than about 80% homologous. A sequence oflabel regions of two or more molecular identifier labels of a set ofmolecular identifier labels may be less than about 60% homologous. Asequence of label regions of two or more molecular identifier labels ofa set of molecular identifier labels may be less than about 40%homologous. A sequence of label regions of two or more molecularidentifier labels of a set of molecular identifier labels may be lessthan about 20% homologous. A label region may be referred to as acellular label region.

The kits and methods disclosed herein may further comprise one or moreprimers. The one or more primers may comprise a sequence that is atleast partially complementary to the universal PCR region. The one ormore primers may comprise a sequence that is at least about 50%complementary to the universal PCR region. The one or more primers maycomprise a sequence that is at least about 80% complementary to theuniversal PCR region.

The kits and methods disclosed herein may further comprise one or moreamplification agents. The amplification agents may comprise a fixedpanel of primers. The amplification agents may comprise one or morecustom primers. The amplification agents may comprise one or morecontrol primers. The amplification agents may comprise one or morehousekeeping gene primers. The amplification agents may comprise one ormore PCR reagents. The one or more PCR reagents may comprisepolymerases, deoxyribonucleotide triphosphates (dNTPs), buffers, or acombination thereof.

The kits and methods disclosed herein may further comprise one or morebeads. The molecular barcodes may be attached to the one or more beads.The sample tags may be attached to the one or more beads. The molecularidentifier labels may be attached to the one or more beads.

Further disclosed herein are methods for generating one or more sets ofbeads. The method may comprise: a) depositing a plurality of firstnucleic acids into a plurality of wells, wherein two or more differentwells of the plurality of wells may comprise two or more differentnucleic acids of the plurality of nucleic acids; b) contacting one ormore wells of the plurality of wells with one or fewer beads to producea plurality of single label beads, wherein a single label bead of theplurality of first labeled beads comprises a bead attached to a nucleicacid of the plurality of first nucleic acids; c) pooling the pluralityof first labeled beads from the plurality of wells to produce a pool offirst labeled beads; d) distributing the pool of first labeled beads toa subsequent plurality of wells, wherein two or more wells of thesubsequent plurality of wells comprise two or more different nucleicacids of a plurality of subsequent nucleic acids; and e) attaching oneor more nucleic acids of the plurality of subsequent nucleic acids toone or more first labeled beads to produce a plurality of uniquelylabeled beads.

The methods and kits disclosed herein may be used to analyze a pluralityof nucleic acids. The methods and kits disclosed herein may be used toanalyze less than about 100,000,000 nucleic acids. The methods and kitsdisclosed herein may be used to analyze less than about 10,000,000nucleic acids. The methods and kits disclosed herein may be used toanalyze less than about 1,000,000 nucleic acids. Further disclosedherein are methods of analyzing a plurality of proteins. The method maycomprise: a) producing a plurality of sample-tagged polypeptides by: i)contacting a first sample comprising a plurality of polypeptides with aplurality of first sample tags to produce a plurality of firstsample-tagged polypeptides; and ii) contacting a second samplecomprising a plurality of polypeptides with a plurality of second sampletags to produce a plurality of second sample-tagged polypeptides,wherein the plurality of first sample tags are different from theplurality of second sample tags; b) contacting the plurality ofsample-tagged polypeptides with a plurality of molecular identifierlabels to produce a plurality of labeled polypeptides; and c) detectingat least a portion of the labeled polypeptides, thereby determining acount of the plurality of polypeptides in the plurality of samples.

The methods of analyzing polypeptides in a plurality of samples mayfurther comprise determining the identity of one or more labeledpolypeptides. Determining the identity of the one or more labeledpolypeptides may comprise mass spectrometry. The method may furthercomprise combining the labeled polypeptides of the first sample with thelabeled polypeptides of the second sample. The labeled polypeptides maybe combined prior to determining the number of different labeledpolypeptides. The method may further comprise combining the firstsample-tagged polypeptides and the second sample-tagged polypeptides.The first sample-tagged polypeptides and the second sample-taggedpolypeptides may be combined prior to contact with the plurality ofmolecular identifier labels. Determining the number of different labeledpolypeptides may comprise detecting at least a portion of the taggedlabeled polypeptide. Detecting at least a portion of the tagged labeledpolypeptide may comprise detecting at least a portion of the sample tag,molecule-specific tag, polypeptide, or a combination thereof.

The methods disclosed herein may comprise contacting a plurality ofsamples with a plurality of sample tags and a plurality of molecularidentifier labels. Contacting the plurality of samples with theplurality of sample tags and the plurality of molecular identifierlabels may occur simultaneously. Contacting the plurality of sampleswith the plurality of sample tags and the plurality of molecularidentifier labels may occur concurrently. Contacting the plurality ofsamples with the plurality of sample tags and the plurality of molecularidentifier labels may occur sequentially. Contacting the plurality ofsamples with the plurality of sample tags may occur prior to contactingthe plurality of samples with the plurality of molecular identifierlabels. Contacting the plurality of samples with the plurality of sampletags may occur after contacting the plurality of samples with theplurality of molecular identifier labels.

The methods disclosed herein may comprise contacting a first sample witha first plurality of sample tags and a first plurality of molecularidentifier labels. Contacting the first sample with the first pluralityof sample tags and the first plurality of molecular identifier labelsmay occur simultaneously. Contacting the first sample with the firstplurality of sample tags and the first plurality of molecular identifierlabels may occur concurrently. Contacting the first sample with thefirst plurality of sample tags and the first plurality of molecularidentifier labels may occur sequentially. Contacting the first samplewith the first plurality of sample tags may occur prior to contactingthe first sample with the first plurality of molecular identifierlabels. Contacting the first sample with the first plurality of sampletags may occur after contacting the first sample with the firstplurality of molecular identifier labels.

The methods disclosed herein may comprise contacting a second samplewith a second plurality of sample tags and a second plurality ofmolecular identifier labels. Contacting the second sample with thesecond plurality of sample tags and the second plurality of molecularidentifier labels may occur simultaneously. Contacting the second samplewith the second plurality of sample tags and the second plurality ofmolecular identifier labels may occur concurrently. Contacting thesecond sample with the second plurality of sample tags and the secondplurality of molecular identifier labels may occur sequentially.Contacting the second sample with the second plurality of sample tagsmay occur prior to contacting the second sample with the secondplurality of molecular identifier labels. Contacting the second samplewith the second plurality of sample tags may occur after contacting thesecond sample with the second plurality of molecular identifier labels.

The methods and kits disclosed herein may further comprise combining twoor more samples. The methods and kits disclosed herein may furthercomprise combining the first sample and the second sample. The first andsecond samples may be combined prior to contact with the plurality ofmolecular identifier labels. The first and second samples may becombined prior to detecting the labeled nucleic acids. The two or moresamples may be combined prior to stochastically labeling two or moremolecules in the two or more samples. The two or more samples may becombined after stochastically labeling two or more molecules in the twoor more samples. The two or more samples may be combined prior todetecting two or more molecules in the two or more samples. The two ormore samples may be combined after detecting two or more molecules inthe two or more samples. The two or more samples may be combined priorto analyzing two or more molecules in the two or more samples. The twoor more samples may be combined after analyzing two or more molecules inthe two or more samples. The two or more samples may be combined priorto conducting one or more assays on two or more molecules in the two ormore samples. The two or more samples may be combined after conductingone or more assays on two or more molecules in the two or more samples.

The methods and kits disclosed herein may comprise conducting one ormore assays on two or more molecules in a sample. The one or more assaysmay comprise one or more amplification reactions. The methods and kitsdisclosed herein may further comprise conducting one or moreamplification reactions to produce labeled nucleic acid amplicons. Thelabeled nucleic acids may be amplified prior to detecting the labelednucleic acids. The method may further comprise combining the first andsecond samples prior to conducting the one or more amplificationreactions.

The amplification reactions may comprise amplifying at least a portionof the sample tag. The amplification reactions may comprise amplifyingat least a portion of the label. The amplification reactions maycomprise amplifying at least a portion of the sample tag, label, nucleicacid, or a combination thereof. The amplification reactions may compriseamplifying at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of thetotal number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying at least about 1% of thetotal number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying at least about 5% of thetotal number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying at least about 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of the total number of labeled nucleic acidsof the plurality of labeled nucleic acids. The amplification reactionsmay comprise amplifying at least about 1% of the total number of labelednucleic acids of the plurality of labeled nucleic acids. Theamplification reactions may comprise amplifying at least about 5% of thetotal number of labeled nucleic acids of the plurality of labelednucleic acids. The amplification reactions may comprise amplifying atleast about 10% of the total number of labeled nucleic acids of theplurality of labeled nucleic acids. The amplification reactions maycomprise amplifying less than about 95%, 90%, 80%, 70%, 60% or 50% ofthe total number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying less than about 50% ofthe total number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying less than about 20% ofthe total number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying less than about 10% ofthe total number of nucleic acids of the plurality of nucleic acids. Theamplification reactions may comprise amplifying less than about 95%,90%, 80%, 70%, 60% or 50% of the total number of labeled nucleic acidsof the plurality of labeled nucleic acids. The amplification reactionsmay comprise amplifying less than about 40% of the total number oflabeled nucleic acids of the plurality of labeled nucleic acids. Theamplification reactions may comprise amplifying less than about 25% ofthe total number of labeled nucleic acids of the plurality of labelednucleic acids. The amplification reactions may comprise amplifying lessthan about 10% of the total number of labeled nucleic acids of theplurality of labeled nucleic acids.

The one or more amplification reactions may result in amplification ofabout 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000targeted nucleic acids in a sample. The one or more amplificationreactions may result in amplification of about 2000 targeted nucleicacids in a sample. The one or more amplification reactions may result inamplification of about 1000 targeted nucleic acids in a sample. The oneor more amplification reactions may result in amplification of about2000 targeted molecules. The one or more amplification reactions mayresult in amplification of about 100 targeted nucleic acids in a sample.

The amplification reactions may comprise one or more polymerase chainreactions (PCRs). The one or more polymerase chain reactions maycomprise multiplex PCR, nested PCR, absolute PCR, HD-PCR, Next Gen PCR,digital RTA, or any combination thereof. The one or more polymerasechain reactions may comprise multiplex PCR. The one or more polymerasechain reactions may comprise nested PCR.

Conducting the one or more amplification reactions may comprise the useof one or more primers. The one or more primers may comprise one or moreoligonucleotides. The one or more oligonucleotides may comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides may compriseless than 12-15 nucleotides. The one or more primers may anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers may anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers may anneal to an internalregion of the plurality of labeled nucleic acids. The internal regionmay be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theinternal region may be at least about 2000 nucleotides from the 3′ endsthe plurality of labeled nucleic acids. The one or more primers maycomprise a fixed panel of primers. The one or more primers may compriseat least one or more custom primers. The one or more primers maycomprise at least one or more control primers. The one or more primersmay comprise at least one or more housekeeping gene primers. The one ormore oligonucleotides may comprise a sequence selected from a groupconsisting of sequences in Table 1. The one or more primers may comprisea universal primer. The universal primer may anneal to a universalprimer binding site. The universal primer may anneal to a universal PCRregion. The one or more custom primers may anneal to at least a portionof a sample tag. The one or more custom primers may anneal to at least aportion of a molecular identifier label. The one or more custom primersmay anneal to at least a portion of a molecular barcode. The one or morecustom primers may anneal to the first sample tag, the second sampletag, the molecular identifier label, the nucleic acid or a productthereof. The one or more primers may comprise a universal primer and acustom primer. The one or more primers may comprise at least about 96 ormore custom primers. The one or more primers may comprise at least about960 or more custom primers. The one or more primers may comprise atleast about 9600 or more custom primers. The one or more custom primersmay anneal to two or more different labeled nucleic acids. The two ormore different labeled nucleic acids may correspond to one or moregenes.

Multiplex PCR reactions may comprise a nested PCR reaction. The nestedPCR reaction may comprise a pair of primers comprising a first primerand a second primer. The first primer may anneal to a region of one ormore nucleic acids of the plurality of nucleic acids. The region of theone or more nucleic acids may be at least about 300 to 400 nucleotidesfrom the 3′ end of the one or more nucleic acids. The second primer mayanneal to a region of one or more nucleic acids of the plurality ofnucleic acids. The region of the one or more nucleic acids may be atleast 200 to 300 nucleotides from the 3′ end of the one or more nucleicacids.

The methods and kits disclosed herein may further comprise conductingone or more cDNA synthesis reactions to produce one or more cDNA copiesof the molecules or derivatives thereof (e.g., labeled molecules). Theone or more cDNA synthesis reactions may comprise one or more reversetranscription reactions.

The methods and kits disclosed herein may comprise one or more samples.The methods and kits disclosed herein may comprise a plurality ofsamples. The plurality of samples may comprise at least about 2, 3, 4,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more samples. Theplurality of samples may comprise at least about 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 or more samples. The plurality ofsamples may comprise at least about 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000 samples, 9000, or 10,000 samples, or 100,000 samples, or1,000,000 or more samples. The plurality of samples may comprise atleast about 10,000 samples. The plurality of samples may comprise atleast about 2 samples. The plurality of samples may comprise at leastabout 5 samples. The plurality of samples may comprise at least about 10samples. The plurality of samples may comprise at least about 50samples. The plurality of samples may comprise at least about 100samples.

The methods and kits disclosed herein may comprise one or more samplescomprising one or more cells. The methods and kits disclosed herein maycomprise two or more samples comprising one or more cells. A firstsample may comprise one or more cells. A second sample may comprise oneor more cells. The one or more cells of the first sample may be of thesame cell type as the one or more cells of the second sample.

The methods and kits disclosed herein may comprise a plurality ofsamples. The plurality of samples may be from one or more subjects. Theplurality of samples may be from two or more subjects. The plurality ofsamples may be from the same subject. The two or more subjects may befrom the same species. The two or more subjects may be from differentspecies. The plurality of samples may be from one or more sources. Theplurality of samples may be from two or more sources. The plurality ofsamples may be from the same subject. The two or more sources may befrom the same species. The two or more sources may be from differentspecies.

The plurality of samples may be obtained concurrently. The plurality ofsamples may be obtained sequentially. The plurality of samples may beobtained over two or more time periods. The two or more time periods maybe one or more hours apart. The two or more time periods may be one ormore days apart. The two or more time periods may be one or more weeksapart. The two or more time periods may be one or more months apart. Thetwo or more time periods may be one or more years apart.

The plurality of samples may be from one or more bodily fluids, tissues,cells, organs, or muscles. The plurality of samples may comprise one ormore blood samples.

The methods and kits disclosed herein may comprise one or more samplescomprising one or more nucleic acids. Two or more samples may compriseone or more nucleic acids. Two or more samples may comprise two or morenucleic acids. The one or more nucleic acids of a first sample may bedifferent from one or more nucleic acids of a second sample. The nucleicacids in a first sample may be at least about 50% identical to thenucleic acids in a second sample. The nucleic acids in a first samplemay be at least about 70% identical to the nucleic acids in a secondsample. The nucleic acids in a first sample may be at least about 80%identical to the nucleic acids in a second sample.

The plurality of nucleic acids in the one or more samples may comprisetwo or more identical sequences. At least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% of the total nucleic acids in the one or more samples may comprisethe same sequence. The plurality of nucleic acids in one or more samplesmay comprise at least two different sequences. At least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100% of the total nucleic acids in the one or moresamples may comprise different sequences.

The plurality of nucleic acids may comprise RNA, DNA, cDNA, mRNA,genomic DNA, small RNA, non-coding RNA, or other nucleic acid contentsof a cell. The plurality of nucleic acids may comprise mRNA. Theplurality of nucleic acids may comprise RNA. The plurality of nucleicacids may comprise mRNA. The plurality of nucleic acids may compriseDNA.

The methods and kits disclosed herein may comprise one or more sampletags. The methods and kits disclosed herein may comprise one or morepluralities of sample tags. The sample tags may comprise a sample indexregion. The sample index region of a first plurality of sample tags maybe different from the sample index region of a second plurality ofsample tags. The sample tags may comprise one or more nucleotides.

The sample tags may comprise at least about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. Thesample tags may comprise at least about 5 or more nucleotides. Thesample tags may comprise at least about 10 or more nucleotides. Thesample tags may comprise less than about 200 nucleotides. The sampletags may comprise less than about 100 nucleotides. The sample tags maycomprise less than about 60 nucleotides.

The sample tags may further comprise a universal primer binding site.The sample tags may further comprise a universal PCR region. The sampletags may further comprise one or more adaptor regions. The sample tagsmay further comprise one or more target-specific regions.

The methods and kits disclosed herein may comprise one or more molecularidentifier labels. The methods and kits disclosed herein may compriseone or more pluralities of molecular identifier labels. The one or morepluralities of molecular identifier labels may comprise two or moredifferent molecular identifier labels. The one or more pluralities ofmolecular identifier labels may comprise 50 or more different molecularidentifier labels. The one or more pluralities of molecular identifierlabels may comprise 90 or more different molecular identifier labels.The one or more pluralities of molecular identifier labels may comprise100 or more different molecular identifier labels. The one or morepluralities of molecular identifier labels may comprise 300 or moredifferent molecular identifier labels. The one or more pluralities ofmolecular identifier labels may comprise 500 or more different molecularidentifier labels. The one or more pluralities of molecular identifierlabels may comprise 960 or more different molecular identifier labels.The one or more pluralities of molecular identifier labels may comprisemultiple copies of one or more molecular identifier labels. Two or morepluralities of molecular identifier labels may comprise one or moreidentical molecular identifier labels. Two or more pluralities ofmolecular identifier labels may comprise 10 or more identical molecularidentifier labels. The molecular identifier labels of a first pluralityof molecular identifier labels may be at least about 30% identical tothe molecular identifier labels of a second plurality of molecularidentifier labels. The molecular identifier labels of a first pluralityof molecular identifier labels may be at least about 50% identical tothe molecular identifier labels of a second plurality of molecularidentifier labels. The molecular identifier labels of a first pluralityof molecular identifier labels may be at least about 80% identical tothe molecular identifier labels of a second plurality of molecularidentifier labels.

The molecular identifier labels may comprise a label region (e.g.,molecular label region, molecular index region). The label region of twoor more molecular identifier labels of a first plurality of molecularidentifier labels may be different. One or more pluralities of molecularidentifier labels may comprise at least about 20 different labelregions. One or more pluralities of molecular identifier labels maycomprise at least about 50 different label regions. One or morepluralities of molecular identifier labels may comprise at least about96 different label regions. One or more pluralities of molecularidentifier labels may comprise at least about 200 different labelregions. One or more pluralities of molecular identifier labels maycomprise at least about 500 different label regions. One or morepluralities of molecular identifier labels may comprise at least about960 different label regions.

The molecular identifier labels may comprise at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more nucleotides. The molecular identifier labelsmay comprise at least about 20, 30, 40, 50 or more nucleotides. Themolecular identifier labels may comprise at least about 21 nucleotides.

The molecular identifier labels may further comprise a target-specificregion. The target-specific region may comprise an oligodT sequence.

The molecular identifier labels may further comprise one or more dyelabels. The molecular identifier labels may further comprise a Cy3 dye.The molecular identifier labels may further comprise a Tye563 dye.

The methods and kits disclosed herein may comprise one or more labeledmolecules. The one or more labeled molecules may be produced bycontacting a plurality of molecules with a plurality of sample tags. Theone or more labeled molecules may be produced by contacting a pluralityof nucleic acids with a plurality of sample tags. Contacting theplurality of nucleic acids with the plurality of sample tags maycomprise ligating one or more sample tags to one or more nucleic acids.Contacting the plurality of nucleic acids with the plurality of sampletag may comprise hybridizing one or more sample tags to one or morenucleic acids. Contacting the plurality of nucleic acids with theplurality of sample tag may comprise performing one or more nucleic acidextension reactions. The one or more nucleic acid extension reactionsmay comprise reverse transcription.

The methods and kits disclosed herein may further comprise attaching oneor more oligonucleotide linkers to the plurality of nucleic acids. Themethod and kits may further comprise attaching one or moreoligonucleotide linkers to the sample tagged nucleic acids. The methodsand kits may further comprise attaching one or more oligonucleotidelinkers to the labeled nucleic acids. The one or more linkers maycomprise at least about 10 nucleotides.

The methods and kits disclosed herein may further comprise attaching oneor more labeled nucleic acids to a support. The support may comprise asolid support. The support may comprise a bead. The support may comprisean array. The support may comprise a glass slide.

Attachment of the labeled nucleic acids to the support may compriseamine-thiol crosslinking, maleimide crosslinking, N-hydroxysuccinimideor N-hydroxysulfosuccinimide, Zenon, SiteClick, or a combinationthereof. Attaching the labeled nucleic acids to the support may compriseattaching biotin to the one or more labeled nucleic acids.

The support may comprise one or more beads. The one or more beads may bea coated bead. The coated bead may be coated with streptavadin.

The support may comprise an array. The array may comprise one or moreprobes. The labeled nucleic acids may be attached to the one or moreprobes. The one or more probes may comprise one or moreoligonucleotides. The one or more probes may be attached to at least aportion of the labeled nucleic acids. The portion of the labeled nucleicacids attached to the one or more probes may comprise at least a portionof the sample tag, molecular identifier label, molecular barcode,nucleic acid, or a combination thereof.

The support may comprise a glass slide. The glass slide may comprise oneor more wells. The one or more wells may be etched on the glass slide.The one or more wells may comprise at least 960 wells. The glass slidemay comprise one or more probes. The one or more probes may be printedonto the glass slide. The one or more wells may further comprise one ormore probes. The one or more probes may be printed within the one ormore wells. The one or more probes may comprise 960 nucleic acids. Thenucleic acids may be different. The nucleic acids may be the same.

The methods and kits disclosed herein may be used to determine a countof one or more molecules in one or more samples. Determining the countof one or more molecules may comprise determining the number ofdifferent labeled nucleic acids. Determining the number of differentlabeled nucleic acids may comprise detecting at least a portion of thelabeled nucleic acid. Detecting at least a portion of the labelednucleic acid may comprise detecting at least a portion of the sampletag, molecular identifier label, molecular barcode, nucleic acid, or acombination thereof.

Determining the number of different labeled nucleic acids may comprisesequencing. Sequencing may comprise MiSeq sequencing. Sequencing maycomprise HiSeq sequencing. Determining the number of different labelednucleic acids may comprise an array. Determining the number of differentlabeled nucleic acids may comprise contacting the labeled nucleic acidswith the one or more probes.

Determining the number of different labeled nucleic acids may comprisecontacting the labeled nucleic acids with an array. The array maycomprise a plurality of probes. Determining the number of differentlabeled nucleic acids may comprise contacting the labeled nucleic acidswith a glass slide of a plurality of probes.

Determining the number of different labeled nucleic acids may compriselabeled probe hybridization, target-specific amplification,target-specific sequencing, sequencing with labeled nucleotides specificfor target small nucleotide polymorphism, sequencing with labelednucleotides specific for restriction enzyme digest patterns, sequencingwith labeled nucleotides specific for mutations, or a combinationthereof.

Determining the number of different labeled nucleic acids may compriseflow cytometry sorting of a sequence-specific label. Determining thenumber of different labeled nucleic acids may comprise detection of thelabeled nucleic acids attached to the beads. Detection of the labelednucleic acids attached to the beads may comprise fluorescence detection.

Determining the number of different labeled nucleic acids may comprisecounting the plurality of labeled nucleic acids by fluorescenceresonance energy transfer (FRET), between a target-specific probe and alabeled nucleic acid or a target-specific labeled probe. Determining thenumber of different labeled nucleic acids may comprise attaching thelabeled nucleic acid to the support.

The methods and kits disclosed herein may further compriseimmunoprecipitation of a target sequence with a nucleic-acid bindingprotein.

The methods and kits disclosed herein may further comprise distributingthe plurality of samples into a plurality of wells of a microwell plate.One or more of the plurality of samples may comprise a plurality ofcells. One or more of the plurality of samples may comprise a pluralityof nucleic acids. The methods and kits disclosed herein may furthercomprise distributing one or fewer cells to the plurality of wells. Theplurality of cells may be lysed in the microwell plate. The methods andkits disclosed herein may further comprise synthesizing cDNA in themicrowell plate. Synthesizing cDNA may comprise reverse transcription ofmRNA.

The methods and kits disclosed herein may further comprise distributingthe plurality of first sample tags, the plurality of second sample tags,the plurality of molecular identifier labels, or any combination thereofinto a microwell plate.

The methods and kits disclosed herein may further comprise distributingone or more beads in the microwell plate. The microwell plate maycomprise a microwell plate fabricated on PDMS by soft lithography,etched on a silicon wafer, etched on a glass slide, patternedphotoresist on a glass slide, or a combination thereof. The microwellmay comprise a hole on a microcapillary plate. The microwell plate maycomprise a water-in-oil emulsion. The microwell plate may comprise atleast one or more wells. The microwell plate may comprise at least about6 wells, 12 wells, 48 wells, 96 wells, 384 wells, 960 wells or 1000wells.

The methods and kits disclosed herein may further comprise a chip. Themicrowell plate may be attached to the chip. The chip may comprise atleast about 6 wells, 12 wells, 48 wells, 96 wells, 384 wells, 960 wells,1000 wells, 2000 wells, 3000 wells, 4000 wells, 5000 wells, 6000 wells,7000 wells, 8000 wells, 9000 wells, 10,000 wells, 20,000 wells, 30,000wells, 40,000 wells, 50,000 wells, 60,000 wells, 70,000 wells, 80,000wells, 90,000 wells, 100,000 wells, 200,000 wells, 500,000 wells, or amillion wells. The wells may comprise an area of at least about 300microns², 400 microns², 500 microns², 600 microns², 700 microns², 800microns², 900 microns², 1000 microns², 1100 microns², 1200 microns²,1300 microns², 1400 microns², 1500 microns². The methods and kitsdisclosed herein may further comprise distributing between about 10,000and 30,000 samples on the chip.

The methods and kits disclosed herein may further comprise diagnosing acondition, disease, or disorder in a subject in need thereof.

The methods and kits disclosed herein may further comprise prognosing acondition, disease, or disorder in a subject in need thereof. Themethods and kits disclosed herein may further comprise determining atreatment for a condition, disease, or disorder in a subject in needthereof.

The plurality of samples may comprise one or more samples from a subjectsuffering from a disease or condition. The plurality of samples maycomprise one or more samples from a healthy subject.

Further disclosed herein is a method of forensic analysis comprising: a)stochastically labeling two or more molecules in two or more samples toproduce two or more labeled molecules; and b) detecting the two or morelabeled molecules.

The method of selecting the custom primer may further comprise selectingthe custom primer based on one or more nucleic acids. The one or morenucleic acids may comprise mRNA transcripts, non-coding transcriptsincluding structural RNAs, transcribed pseudogenes, model mRNA providedby a genome annotation process, sequences corresponding to a genomiccontig, or any combination thereof. The one or more nucleic acids may beRNA. The one or more nucleic acids may be mRNA. The one or more nucleicacids may comprise one or more exons. The method of selecting the customprimer may further comprise enriching for one or more subsets of nucleicacids. The one or more subsets comprise low abundance mRNAs. The methodof selecting the custom primer may further comprise a computationalalgorithm.

The methods and kits disclosed herein may comprise the use of one ormore controls. The one or more controls may be spiked in controls. Theone or more controls may comprise nucleic acids. The one or more samplescomprising a plurality of nucleic acids may be spiked with one or morecontrol nucleic acids. The one or more control nucleic acids may be usedto measure an efficiency of producing the labeled nucleic acid library.

The methods and kits disclosed herein may be used in the production ofone or more nucleic acid libraries. The one or more nucleic acidlibraries may comprise a plurality of labeled nucleic acids orderivatives thereof (e.g., labeled amplicons). The method of producingthe labeled nucleic acid library may comprise stochastically labelingtwo or more nucleic acids in two or more samples with two or more setsof molecular barcodes to produce a plurality of labeled nucleic acids.The method of producing a labeled nucleic acid library may comprisecontacting two or more samples with a plurality of sample tags and aplurality of molecule specific labels to produce a plurality of labelednucleic acids. The labeled nucleic acids may comprise a sample indexregion, a label region and a nucleic acid region. The sample indexregion may be used to confer a sample or sub-sample identity to thenucleic acid. The sample index region may be used to determine thesource of the nucleic acid. The label region may be used to confer aunique identity to the nucleic acid, thereby enabling differentiation oftwo or more identical nucleic acids in the same sample or sub-sample.

The method of producing a nucleic acid library may further compriseamplifying one or more labeled nucleic acids to produce one or moreenriched labeled nucleic acids. The method may further compriseconducting one or more pull-down assays of the one or more enrichedlabeled nucleic acids. The method may further comprise purifying the oneor more enriched labeled nucleic acids.

The kits disclosed herein may comprise a plurality of beads, a primerand/or amplification agents. One or more kits may be used in theanalysis of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ormore samples or sub-samples. One or more kits may be used in theanalysis of at least about 96 samples. One or more kits may be used inthe analysis of at least about 384 samples. The kit may further compriseinstructions for primer design and optimization.

The kit may further comprise one or more microwell plates. The one ormore microwell plates may be used for the distribution of one or morebeads. The one or more microwell plates may be used for the distributionof one or more molecules or derivatives thereof (e.g., labeledmolecules, labeled amplicons) from one or more samples.

The kit may further comprise one or more additional containers. The oneor more additional containers may comprise one or more additionalpluralities of sample tags. The one or more additional pluralities ofsample tags in the one or more additional containers may be differentfrom the first plurality of sample tags in the first container. The oneor more additional containers may comprise one or more additionalpluralities of molecular identifier labels. The one or more additionalpluralities of molecular identifier labels of the one or more additionalcontainers may be at least about 50% identical to the one or moreadditional molecular identifier labels of the second container. The oneor more additional pluralities of molecular identifier labels of the oneor more additional containers may be at least about 80% identical to theone or more additional molecular identifier labels of the secondcontainer. The one or more additional pluralities of molecularidentifier labels of the one or more additional containers may be atleast about 90% identical to the one or more additional molecularidentifier labels of the second container.

Further disclosed herein are methods of producing one or more sets oflabeled beads. The method of producing the one or more sets of labeledbeads may comprise attaching one or more nucleic acids to one or morebeads, thereby producing one or more sets of labeled beads. The one ormore nucleic acids may comprise one or more molecular barcodes. The oneor more nucleic acids may comprise one or more sample tags. The one ormore nucleic acids may comprise one or more molecular identifier labels.The one or more nucleic acids may comprise a) a primer region; b) asample index region; and c) a linker or adaptor region. The one or morenucleic acids may comprise a) a primer region; b) a label region; and c)a linker or adaptor region. The one or more nucleic acids may comprisea) a sample index region; and b) a label region. The one or more nucleicacids may further comprise a primer region. The one or more nucleicacids may further comprise a target specific region. The one or morenucleic acids may further comprise a linker region. The one or morenucleic acids may further comprise an adaptor region. The one or morenucleic acids may further comprise a sample index region. The one ormore nucleic acids may further comprise a label region.

The primer region of the nucleic acids for a set of labeled beads may beat least about 70% identical. The primer region of the nucleic acids fora set of labeled beads may be at least about 90% identical. The primerregion of the nucleic acids for a set of labeled beads may be the same.

The sample index region of the nucleic acids for a set of labeled beadsmay be at least about 70% identical. The sample index region of thenucleic acids for a set of labeled beads may be at least about 90%identical. The sample index region of the nucleic acids for a set oflabeled beads may be the same. The sample index region of the nucleicacids for two or more sets of sample indexed beads may be less thanabout 40% identical. The sample index region of the nucleic acids fortwo or more sets of sample indexed beads may be less than about 50%identical. The sample index region of the nucleic acids for two or moresets of sample indexed beads may be less than about 60% identical. Thesample index region of nucleic acids for two or more sets of sampleindexed beads may be different.

The label region of the nucleic acids for two or more sets of labeledbeads may be at least about 70% identical. The label region of thenucleic acids for two or more sets of labeled beads may be at leastabout 90% identical. The label region of the nucleic acids for two ormore sets of labeled beads may be the same. The label region of thenucleic acids for a set of labeled beads may be less than about 40%identical. The label region of the nucleic acids for a set of labeledbeads may be less than about 50% identical. The label region of thenucleic acids for a set of labeled beads may be less than about 60%identical. The label region of two or more nucleic acids for a set oflabeled beads may be different.

The linker or adaptor region of the nucleic acids for a set of labeledbeads may be at least about 70% identical. The linker or adaptor regionof the nucleic acids for a set of labeled beads may be at least about90% identical. The linker or adaptor region of the nucleic acids for aset of labeled beads may be the same.

The target specific region of the nucleic acids for two or more sets oftarget specified beads may be at least about 70% identical. The targetspecific region of the nucleic acids for two or more sets of targetspecified beads may be at least about 90% identical. The target specificregion of the nucleic acids for two or more sets of target specifiedbeads may be the same. The target specific region of the nucleic acidsfor a set of target specified beads may be less than about 40%identical. The target specific region of the nucleic acids for a set oftarget specified beads may be less than about 50% identical. The targetspecific region of the nucleic acids for a set of target specified beadsmay be less than about 60% identical. The target specific region of twoor more nucleic acids for a set of target specified beads may bedifferent.

The one or more sets of labeled beads may comprise one million or morelabeled beads. The one or more sets of labeled beads may comprise tenmillion or more labeled beads.

Attaching the one or more nucleic acids to the beads may comprisecovalent attachment. Attaching the one or more nucleic acids to thebeads may comprise conjugation. Attaching the one or more nucleic acidsto the beads may comprise ionic interactions.

The beads may be coated beads. The nucleic acids may be attached to oneor more tags. The beads may be coated with streptavidin. The nucleicacids may be attached to biotin. The beads may also be coated withantibodies or nucleic acids, and the nucleic acids may be attached tothe beads indirectly via such surface coated materials.

In one aspect, the disclosure provides for a composition comprising: asolid support, wherein said solid support comprises a plurality ofoligonucleotides, wherein at least two of said plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein said cellular labels of said at least two of said plurality ofoligonucleotides are the same, and wherein said molecular labels of saidat least two of said plurality of oligonucleotides are different. Insome embodiments, the plurality of oligonucleotide further comprises asample label. In some embodiments, the plurality of oligonucleotidesfurther comprises a target binding region. In some embodiments, thetarget binding region comprises a sequence is adapted to hybridize to atarget nucleic acid. In some embodiments, the target binding regioncomprises a sequence selected from the group consisting of: a randommultimer e.g., a random dimer, trimer, quatramer, pentamer, hexamer,septamer, octamer, nonamer, decamer, or higher multimer sequence of anylength; a gene-specific primer; and oligo dT; or any combinationthereof. In some embodiments, the plurality of oligonucleotidescomprises a universal label. In some embodiments, the universal labelcomprises a binding site for a sequencing primer. In some embodiments,the plurality of oligonucleotides comprises a linker. In someembodiments, the linker comprises a functional group. In someembodiments, the linker is located 5′ to said oligonucleotide. In someembodiments, the linker is selected from the group consisting of: C6,biotin, streptavidin, primary amines, aldehydes, and ketones, or anycombination thereof. In some embodiments, the solid support is comprisedof polystyrene. In some embodiments, the in solid support is magnetic.In some embodiments, the solid support is selected from the groupconsisting of: a PDMS solid support, a glass solid support, apolypropylene solid support, an agarose solid support, a gelatin solidsupport, a magnetic solid support, and a pluronic solid support, or anycombination thereof. In some embodiments, the solid support comprises abead. In some embodiments, the solid support comprises a diameter ofabout 20 microns. In some embodiments, the solid support comprises adiameter from about 5 microns to about 40 microns. In some embodiments,the solid support comprises a functional group. In some embodiments, thefunctional group is selected from the group consisting of: C6, biotin,streptavidin, primary amines, aldehydes, and ketones, or any combinationthereof. In some embodiments, the cellular label comprises a pluralityof cellular labels. In some embodiments, the plurality of cellularlabels are interspersed with a plurality of linker label sequences. Insome embodiments, the plurality of oligonucleotides comprises from10,000 to 1 billion oligonucleotides.

In one aspect the disclosure provides for a solid support comprising: afirst oligonucleotide comprising: a first cellular label comprising afirst random sequence, a second random sequence, and a first linkerlabel sequence, wherein said first linker label sequence connects saidfirst random sequence and said second random sequence; and a firstmolecular label comprising a random sequence; and a secondoligonucleotide comprising: a second cellular label comprising a thirdrandom sequence, a fourth random sequence, and a second linker labelsequence, wherein said second linker label sequence connects said thirdrandom sequence and said fourth random sequence; and a second molecularlabel comprising a random sequence, wherein said first cellular labeland said second cellular label are the same and said first molecularlabel and said second molecular label are different. In someembodiments, the first and second oligonucleotides further compriseidentical sample index regions. In some embodiments, the sample indexregion comprises a random sequence. In some embodiments, the sampleindex region is 4-12 nucleotides in length. In some embodiments, thecellular label is directly attached to said molecular label. In someembodiments, the cellular label and said molecular label are attachedthrough a linker label sequence. In some embodiments, the randomsequence of said cellular label is from 4-12 nucleotides in length. Insome embodiments, the constant sequence of said cellular label is atleast 4 nucleotides in length. In some embodiments, the cellular labelhas a total length of at least 12 nucleotides. In some embodiments, thecellular label further comprises one or more additional randomsequences. In some embodiments, the cellular label further comprises oneor more additional linker label sequences. In some embodiments, the oneor more additional linker label sequences connect the one or moreadditional random sequences. In some embodiments, the random sequence ofthe molecular label is 4-12 nucleotides in length.

In one aspect the disclosure provides for a composition comprising: asolid support, wherein said solid support comprises a plurality ofoligonucleotides, wherein at least two of said plurality ofoligonucleotides comprises: a cellular label, a molecular label; and atarget binding region; and a plurality of a target nucleic acids,wherein said cellular labels of said at least two of said plurality ofoligonucleotides are the same, and wherein said molecular labels of saidat least two of said plurality of oligonucleotides are different. Insome embodiments, the target binding region comprises a sequence that isadapted to hybridize to at least one of said plurality of target nucleicacids. In some embodiments, the target binding region comprises asequence selected from the group consisting of: a random a randommultimer e.g., a random dimer, trimer, quatramer, pentamer, hexamer,septamer, octamer, nonamer, decamer, or higher multimer sequence of anylength; a gene-specific primer; and oligo dT; or any combinationthereof. In some embodiments, the plurality of oligonucleotidescomprises from 10,000 to 1 billion oligonucleotides. In someembodiments, the plurality of oligonucleotides comprises a number ofoligonucleotides greater than the number of target nucleic acids of saidplurality of target nucleic acids. In some embodiments, the plurality oftarget nucleic acids comprises multiple copies of a same target nucleicacid. In some embodiments, the plurality of target nucleic acidscomprises multiple copies of different target nucleic acids. In someembodiments, the plurality of target nucleic acids are bound to saidplurality of oligonucleotides. In some embodiments, the oligonucleotidefurther comprises a sample label. In some embodiments, the plurality ofoligonucleotides comprises a universal label. In some embodiments, theuniversal label comprises a binding site for a sequencing primer. Insome embodiments, the plurality of oligonucleotides comprises a linker.In some embodiments, the linker comprises a functional group. In someembodiments, the linker is located 5′ to said oligonucleotide. In someembodiments, the functional group comprises an amino group. In someembodiments, the linker is selected from the group consisting of: C6,biotin, streptavidin, primary amines, aldehydes, and ketones, or anycombination thereof. In some embodiments, the solid support is comprisedof polystyrene. In some embodiments, the solid support is magnetic. Insome embodiments, the solid support is selected from the groupconsisting of: a PDMS solid support, a glass solid support, apolypropylene solid support, an agarose solid support, a gelatin solidsupport, a magnetic solid support, and a pluronic solid support, or anycombination thereof. In some embodiments, the solid support comprises abead. In some embodiments, the solid support comprises a diameter ofabout 20 microns. In some embodiments, the solid support comprises adiameter from about 5 microns to about 40 microns. In some embodiments,the solid support comprises a functional group. In some embodiments, thefunctional group comprises a carboxy group. In some embodiments, thefunctional group is selected from the group consisting of: C6, biotin,streptavidin, primary amines, aldehydes, and ketones, or any combinationthereof. In some embodiments, the cellular label comprises a pluralityof cellular labels. In some embodiments, the plurality of cellularlabels is interspersed with a plurality of linker label sequences.

In one aspect the disclosure provides for a kit comprising: a firstsolid support, wherein said first solid support comprises a firstplurality of oligonucleotides, wherein said first plurality ofoligonucleotides comprises the same first cellular label, a second solidsupport, wherein said second solid support comprises a second pluralityof oligonucleotides, wherein said second plurality of oligonucleotidescomprises the same second cellular label, instructions for use, whereinsaid first cellular label and said second cellular label are different.In some embodiments, oligonucleotides form said first plurality ofoligonucleotides and said second plurality of oligonucleotides comprisesa molecular label. In some embodiments, the molecular labels of saidoligonucleotides are different. In some embodiments, the molecularlabels of said oligonucleotides are the same. In some embodiments, themolecular label of some of said oligonucleotides are different and someare the same. In some embodiments, the oligonucleotides from said firstplurality of oligonucleotides and said second plurality ofoligonucleotides comprise a target binding region. In some embodiments,the kit further comprises: a microwell array. In some embodiments, thekit further comprises: a buffer. In some embodiments, the buffer isselected from the group consisting of: a reconstitution buffer, adilution buffer, and a stabilization buffer, or any combination thereof.

In one aspect the disclosure provides for a method for determining anamount of a target nucleic acid comprising: contacting a sample with asolid support, wherein said solid support comprises a plurality ofoligonucleotides, wherein at least two of said plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein said cellular labels of said at least two of said plurality ofoligonucleotides are the same, and wherein said molecular labels of saidat least two of said plurality of oligonucleotides are different; andhybridizing said target nucleic acid from said sample to anoligonucleotide of said plurality of oligonucleotides. In someembodiments, the sample comprises cells. In some embodiments, the sampleis lysed prior to said hybridizing. In some embodiments, the hybridizingcomprising hybridizing multiple copies of a same target nucleic acid tosaid plurality of oligonucleotides. In some embodiments, the methodfurther comprises: amplifying said target nucleic acid. In someembodiments, the amplifying comprises reverse transcribing said targetnucleic acid. In some embodiments, the amplifying comprisesamplification using a method selected from the group consisting of: PCR,quantitative PCR, real-time PCR, and digital PCR, or any combinationthereof. In some embodiments, the amplifying is performed directly onsaid solid support. In some embodiments, the amplifying is performed ona template transcribed from said solid support. In some embodiments, themethod further comprises: sequencing said target nucleic acid. In someembodiments, the sequencing comprises sequencing said target nucleicacid and said molecular label. In some embodiments, the method furthercomprises: determining an amount of said target nucleic acid. In someembodiments, the determining comprises quantifying levels of said targetnucleic acid. In some embodiments, the determining comprises countingthe number of sequenced molecular labels for said target nucleic acid.In some embodiments, the contacting occurs in a microwell. In someembodiments, the microwell is made from a material selected from thegroup consisting of: hydrophilic plastic, plastic, elastomer, andhydrogel, or any combination thereof. In some embodiments, the microwellcomprises agarose. In some embodiments, the microwell is one microwellof a microwell array. In some embodiments, the microwell array comprisesat least 90 microwells. In some embodiments, the microwell arraycomprises at least 150,000 microwells. In some embodiments, themicrowell comprises at least one solid support per well. In someembodiments, the microwell comprises at most two solid supports perwell. In some embodiments, the microwell is of a size that accommodatesat most two of said solid support. In some embodiments, the microwell isof a size that accommodates at most one solid support. In someembodiments, the microwell is at least 25 microns deep. In someembodiments, the microwell is at least 25 microns in diameter.

In one aspect the disclosure provides for a method to reduceamplification bias of a target nucleic acid comprising: contacting asample to a solid support, wherein said solid support comprises aplurality of oligonucleotides, wherein at least two of said plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein said cellular labels of said at least two of said plurality ofoligonucleotides are the same, and wherein said molecular labels of saidat least two of said plurality of oligonucleotides are different; andhybridizing a target nucleic acid from said sample to said plurality ofoligonucleotides; amplifying said target nucleic acid; sequencing saidtarget nucleic acid, wherein said sequencing sequences said targetnucleic acid and said molecular label of said oligonucleotide to whichsaid target nucleic acid is bound; and determining an amount of saidtarget nucleic acid. In some embodiments, the hybridizing comprisinghybridizing multiple copies of a same target nucleic acid to saidplurality of oligonucleotides. In some embodiments, the determiningcomprises counting a number of sequenced molecular labels for a sametarget nucleic acid. In some embodiments, the counting counts the numberof copies of said same target nucleic acid. In some embodiments, thesample comprises cells. In some embodiments, the amplifying comprisesreverse transcribing said target nucleic acid. In some embodiments, theamplifying comprises amplification using a method selected from thegroup consisting of: PCR, quantitative PCR, real-time PCR, and digitalPCR, or any combination thereof. In some embodiments, the amplifying isperformed directly on said solid support. In some embodiments, theamplifying is performed on a template transcribed from said solidsupport.

In one aspect the disclosure provides for a composition comprising: amicrowell; a cell; and a solid support, wherein said solid supportcomprises a plurality of oligonucleotides, wherein at least two of saidplurality of oligonucleotides comprises a cellular label and a molecularlabel, wherein said cellular labels of said at least two of saidplurality of oligonucleotides are the same, and wherein said molecularlabels of said at least two of said plurality of oligonucleotides aredifferent. In some embodiments, the at least two of said plurality ofoligonucleotides further comprises a sample label. In some embodiments,the at least two of said plurality of oligonucleotides further comprisesa target binding region. In some embodiments, the target binding regioncomprises a sequence selected from the group consisting of: a randommultimer e.g., a random dimer, trimer, quatramer, pentamer, hexamer,septamer, octamer, nonamer, decamer, or higher multimer sequence of anylength; a gene-specific primer; and oligo dT; or any combinationthereof. In some embodiments, the plurality of oligonucleotidescomprises a universal label. In some embodiments, the universal labelcomprises a binding site for a sequencing primer. In some embodiments,the solid support is comprised of polystyrene. In some embodiments, thesolid support is magnetic. In some embodiments, the solid support isselected from the group consisting of: a PDMS solid support, a glasssolid support, a polypropylene solid support, an agarose solid support,a gelatin solid support, a magnetic solid support, and a pluronic solidsupport, or any combination thereof. In some embodiments, the solidsupport comprises a bead. In some embodiments, the solid support has adiameter of about 20 microns. In some embodiments, the solid support hasa diameter from about 5 microns to about 40 microns. In someembodiments, the cellular label comprises a plurality of cellularlabels. In some embodiments, the plurality of cellular labels isinterspersed with a plurality of linker sequences. In some embodiments,the microwell is made from a material selected from the group consistingof: hydrophilic plastic, plastic, elastomer, and hydrogel, or anycombination thereof. In some embodiments, the microwell comprisesagarose. In some embodiments, the microwell is a microwell of amicrowell array. In some embodiments, the microwell comprises at leastone solid support per well. In some embodiments, the microwell comprisesat most two solid supports per well. In some embodiments, the microwellis of a size that accommodates at least one of said solid support and atleast one of said cell. In some embodiments, the microwell is of a sizethat accommodates at most one of said solid support and at least one ofsaid cell. In some embodiments, the microwell is at least 25 micronsdeep. In some embodiments, the microwell is at least 25 microns indiameter. In some embodiments, the microwell is flat.

In one aspect the disclosure provides for a device comprising: a firstsubstrate comprising a first microwell array; wherein said firstmicrowell array comprises a plurality of first microwells in a firstpre-determined spatial arrangement configured to perform multiplexed,single cell stochastic labeling and molecular indexing assays.

In some embodiments, the device comprises a first substrate comprisingat least a second microwell array, wherein said at least secondmicrowell array comprises a plurality of at least second microwells inan at least second pre-determined spatial arrangement. In someembodiments, the first microwells and the at least second microwells arethe same. In some embodiments, the first microwells and the at leastsecond microwells are different. In some embodiments, the firstpre-determined spatial arrangement and the at least secondpre-determined spatial arrangement are the same. In some embodiments,the first pre-determined spatial arrangement and the at least secondpre-determined spatial arrangement are different. In some embodiments, apre-determined spatial arrangement comprises a one dimensional or twodimensional array pattern. In some embodiments, the two dimensionalarray pattern comprises a square grid, a rectangular grid, or ahexagonal grid. In some embodiments, the microwells comprise acylindrical geometry, a conical geometry, a hemispherical geometry, arectangular geometry, a polyhedral geometry, or a combination thereof.In some embodiments, a diameter of the microwells is between about 5microns and about 50 microns. In some embodiments, a depth of themicrowells is between about 10 microns and about 60 microns. In someembodiments, a center-to-center spacing between two adjacent microwellsis between about 15 microns and about 75 microns. In some embodiments, atotal number of microwells in a first or at least second microwell arrayis between about 96 and about 5,000,000. In some embodiments, the firstsubstrate comprises silicon, fused-silica, glass, a polymer, a metal, ora combination thereof. In some embodiments, the first substrate furthercomprises agarose or a hydrogel. In some embodiments, a microwell arrayfurther comprises at least one surface feature, wherein said surfacefeature surrounds one or more individual microwells or straddles asurface between individual microwells, and wherein said surface featureis domed, ridged, or peaked.

In one aspect the disclosure provides for a device comprising: a firstsubstrate comprising at least a first microwell array; and a mechanicalfixture comprising a top plate, a bottom plate, and a gasket; whereinwhen the first substrate and mechanical fixture are in assembled form,the first substrate is positioned between the gasket and the bottomplate, the gasket forms a leak-proof seal with the first substrate, andthe top plate and gasket form at least a first chamber encompassing saidat least first microwell array such that a cell sample and a bead-basedoligonucleotide label may be dispensed into said at least first chamberto perform multiplexed, single cell stochastic labeling and a molecularindexing assays.

In some embodiments, the at least first microwell array is any describedherein. In some embodiments, the gasket is fabricated frompolydimethylsiloxane (PDMS) or a similar elastomeric material. In someembodiments, the top and bottom plates are fabricated from aluminum,anodized aluminum, stainless steel, teflon, polymethylmethacrylate,polycarbonate, or a similar rigid polymer material.

In one aspect the disclosure provides for a device comprising: at leastone substrate further comprising at least one microwell array; and aflow cell; wherein the flow cell encloses or is attached to said atleast one substrate, and includes at least one inlet port and at leastone outlet port for the purpose of delivering fluids to said microwellarrays; and wherein the device is configured to perform multiplexed,single cell stochastic labeling and molecular indexing assays.

In some embodiments, said at least one substrate comprise at least onemicrowell array as described herein. In some embodiments, the flow cellfurther comprises a plurality of microarray chambers that interface witha plurality of microwell arrays such that one or more samples may beprocessed in parallel. In some embodiments, the flow cell furthercomprises a porous barrier or flow diffuser to provide more uniformdelivery of cells and beads to the at least one microwell array. In someembodiments, the flow cell further comprises dividers that divide eachchamber containing a microwell array into subsections that collectivelycover the same total array area and provide for more uniform delivery ofcells and beads to the at least one microwell array. In someembodiments, the width of fluid channels incorporated into the device isbetween about 50 microns and 20 mm. In some embodiments, the depth offluid channels incorporated into the device is between about 50 micronsand about 2 mm. In some embodiments, the flow cell is fabricated from amaterial selected from the group consisting of silicon, fused-silica,glass, polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate(PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), highdensity polyethylene (HDPE), polyimide, cyclic olefin polymers (COP),cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxyresin, metal, or a combination of these materials. In some embodiments,the device comprises a fixed component of an instrument systemconfigured to perform automated multiplexed, single cell stochasticlabeling and molecular indexing assays. In some embodiments, the devicecomprises a removable component of an instrument system configured toperform automated multiplexed, single cell stochastic labeling andmolecular indexing assays.

In one aspect the disclosure provides for a cartridge comprising: atleast a first substrate further comprising at least a first microwellarray; at least a first flow cell or microwell array chamber; one ormore sample or reagent reservoirs; and wherein the cartridge furthercomprises at least one inlet port and at least one outlet port for thepurpose of delivering fluids to said at least first microwell array; andwherein the cartridge is configured to perform multiplexed, single cellstochastic labeling and molecular indexing assays.

In some embodiments, said at least first substrate comprises at least afirst microwell array as described herein. In some embodiments, thecartridge comprises a plurality of microwell arrays and is configured toprocess one or more samples in parallel. In some embodiments, the atleast first flow cell or microwell array chamber further comprises aporous barrier or flow diffuser to provide more uniform delivery ofcells and beads to the at least first microwell arrays. In someembodiments, the at least first flow cell or microwell array chamberfurther comprises dividers that divide the at least first flow cell ormicrowell array chamber into subsections that collectively cover thesame total array area and provide for more uniform delivery of cells andbeads to the microwell arrays. In some embodiments, the width of fluidchannels incorporated into the cartridge is between about 50 microns and200 microns. In some embodiments, the width of the fluid channelsincorporated into the cartridge is between about 200 microns and 2 mm.In some embodiments, the width of the fluid channels incorporated intothe cartridge is between about 2 mm and 10 mm. In some embodiments, thewidth of the fluid channels incorporated into the cartridge is betweenabout 10 mm and 20 mm. In some embodiments, the depth of fluid channelsincorporated into the cartridge is between about 50 microns and about 2mm. In some embodiments, the depth of fluid channels incorporated intothe cartridge is between about 500 microns and 1 mm. In someembodiments, the depth of fluid channels incorporated into the cartridgeis between about 1 mm and about 2 mm. In some embodiments, the one ormore flow cells or microwell array chambers are fabricated from amaterial selected from the group consisting of silicon, fused-silica,glass, polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate(PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), highdensity polyethylene (HDPE), polyimide, cyclic olefin polymers (COP),cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxyresin, metal, or a combination of these materials. In some embodiments,the device comprises a removable, consumable component of an instrumentsystem configured to perform automated multiplexed, single cellstochastic labeling and molecular indexing assays. In some embodiments,the cartridge further comprises bypass channels or other design featuresfor providing self-metering of cell samples or bead suspensionsdispensed or injected into the cartridge. In some embodiments, thecartridge further comprises integrated miniature pumps for controllingfluid flow through the device. In some embodiments, the cartridgefurther comprises integrated miniature valves for compartmentalizingpre-loaded reagents and for controlling fluid flow through the device.In some embodiments, the cartridge further comprises vents for providingan escape path for trapped air. In some embodiments, the cartridgefurther comprises design elements for creating physical or chemicalbarriers that effectively increase pathlength and prevent or minimizediffusion of molecules between microwells, wherein the design elementsare selected from the group consisting of: a pattern of serpentinechannels for delivery of cells and beads to the at least first microwellarray, a retractable platen or deformable membrane that is pressed intocontact with the surface of the at least first microwell array, or therelease of an immiscible, hydrophobic fluid from a reservoir within thecartridge. In some embodiments, the cartridge further comprisesintegrated temperature control components or an integrated thermalinterface for providing good thermal contact with an external instrumentsystem. In some embodiments, the cartridge further comprises an opticalinterface or window for optical imaging of the at least first microwellarray. In some embodiments, the cartridge further comprises one or moreremovable sample collection chambers that are configured to interfacewith stand-alone PCR thermal cyclers and/or sequencing instruments. Insome embodiments, the cartridge itself is configured to interfacedirectly with stand-alone PCR thermal cyclers and/or sequencinginstruments.

In one aspect the disclosure provides for an instrument systemcomprising: at least a first flow cell or cartridge further comprisingat least a first microwell array; and a flow controller; wherein theflow controller controls the delivery of cell samples, bead-basedoligonucleotide labeling reagents, and other assay reagents to the atleast first microwell array, and the instrument system is configured toperform multiplexed, single cell stochastic labeling and molecularindexing assays.

In some embodiments, the at least first microwell array as describedherein. In some embodiments, the at least first flow cell is a fixedcomponent of the system. In some embodiments, the at least first flowcell is a removable, consumable component of the system. In someembodiments, the at least first cartridge is a removable, consumablecomponent of the system. In some embodiments, cell samples andbead-based oligonucleotide reagents are dispensed or injected directlyinto the cartridge by the user. In some embodiments, assay reagentsother than cell samples are preloaded in the cartridge. In someembodiments, the instrument system further comprises an imaging systemfor imaging the at least first microwell array. In some embodiments, theinstrument system further comprises a cell or bead distribution systemfor facilitating uniform distribution of cells and beads across the atleast first microwell array, wherein the mechanism underlying saiddistribution system is selected from the group consisting of rocking,shaking, swirling, recirculating flow, low frequency agitation, or highfrequency agitation. In some embodiments, the instrument system furthercomprises a cell lysis system wherein the system uses a high frequencypiezoelectric transducer for sonicating the cells. In some embodiments,the instrument system further comprises a temperature controller formaintaining a user-specified temperature, or for ramping temperaturebetween two or more specified temperatures over two or more specifiedtime intervals. In some embodiments, the instrument system furthercomprises a magnetic field controller for use in eluting beads frommicrowells. In some embodiments, the instrument system further comprisesa computer or processor programmed to provide a user interface andcontrol of system functions. In some embodiments, the instrument systemfurther comprises program code for providing real-time image analysiscapability. In some embodiments, the real-time image analysis andinstrument control functions are coupled, so that cell and bead sampleloading steps can be prolonged or repeated until optimal cell/beaddistributions are achieved. In some embodiments, the instrument systemfurther comprises an integrated PCR thermal cycler for amplification ofoligonucleotide labels. In some embodiments, the instrument systemfurther comprises an integrated sequencer for sequencing ofoligonucleotide libraries, thereby providing sample-to-answercapability. In some embodiments, the cell samples comprise patientsamples and the results of the multiplexed, single cell stochasticlabeling and molecular indexing assay are used for clinical diagnosticapplications. In some embodiments, the cell samples comprise patientsamples and the results of the multiplexed, single cell stochasticlabeling and molecular indexing assay are used by a healthcare providerto make informed healthcare treatment decisions.

In one aspect the disclosure provides for software residing in acomputer readable medium programmed to perform one or more of thefollowing sequence data analysis functions: determining the number ofreads per gene per cell, and the number of unique transcript moleculesper gene per cell; principal component analysis or other statisticalanalysis to predict confidence intervals for determinations of thenumber of transcript molecules per gene per cell; alignment of genesequence data with known reference sequences; decoding/demultiplexing ofsample barcodes, cell barcodes, and molecular barcodes; and automatedclustering of molecular labels to compensate for amplification orsequencing errors; wherein the sequence data is generated by performingmultiplexed, single cell stochastic labeling and molecular indexingassays.

In one aspect the disclosure provides for a composition comprising: asolid support, wherein the solid support comprises a plurality ofoligonucleotides, wherein at least two of the plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein the cellular labels of the at least two of the plurality ofoligonucleotides are the same, and wherein the molecular labels of theat least two of the plurality of oligonucleotides are different.

In some embodiments, the plurality of oligonucleotide further comprisesa sample label. In some embodiments, the plurality of oligonucleotidesfurther comprises a target binding region. In some embodiments, thetarget binding region comprises a sequence is adapted to hybridize to atarget nucleic acid. In some embodiments, the target nucleic acidcomprises a plurality of target nucleic acids comprising at least 0.01%,0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the transcripts of atranscriptome of an organism. In some embodiments, the target nucleicacid is DNA. In some embodiments, the target nucleic acid is RNA. Insome embodiments, the target nucleic acid is mRNA. In some embodiments,the DNA is genomic DNA. In some embodiments, the genomic DNA is sheared.In some embodiments, the sheared genomic DNA comprises at least 0.01%,0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the genes of a genomeof an organism. In some embodiments, the target binding region comprisesa sequence selected from the group consisting of: a random multimere.g., a random dimer, trimer, quatramer, pentamer, hexamer, septamer,octamer, nonamer, decamer, or higher multimer sequence of any length; agene-specific primer; and oligo dT; or any combination thereof. In someembodiments, the plurality of oligonucleotides comprises a universallabel. In some embodiments, the universal label comprises a binding sitefor a sequencing primer. In some embodiments, the plurality ofoligonucleotides comprises a linker. In some embodiments, the linkercomprises a functional group. In some embodiments, the linker is located5′ to the oligonucleotide. In some embodiments, the linker is selectedfrom the group consisting of: C6, biotin, streptavidin, primary amines,aldehydes, and ketones, or any combination thereof. In some embodiments,the solid support is comprised of polystyrene. In some embodiments, insolid support is magnetic. In some embodiments, the solid support isselected from the group consisting of: a PDMS solid support, a glasssolid support, a polypropylene solid support, an agarose solid support,a gelatin solid support, a magnetic solid support, and a pluronic solidsupport, or any combination thereofln some embodiments, the solidsupport comprises a bead. In some embodiments, the solid supportcomprises a diameter of about 20 microns. In some embodiments, the solidsupport comprises a diameter from about 5 microns to about 40 microns.In some embodiments, the solid support comprises a functional group. Insome embodiments, the functional group is selected from the groupconsisting of: C6, biotin, streptavidin, primary amines, aldehydes, andketones, or any combination thereof. In some embodiments, the cellularlabel comprises a plurality of cellular labels. In some embodiments, theplurality of cellular labels is interspersed with a plurality of linkerlabel sequences. In some embodiments, the plurality of oligonucleotidescomprises from 10,000 to 1 billion oligonucleotides. In someembodiments, the plurality of oligonucleotides comprises from 10,000 to1 billion target binding regions. In some embodiments, the plurality ofoligonucleotides comprises from 10,000 to 1 billion different targetbinding regions. In some embodiments, the plurality of oligonucleotidescomprises from 10,000 to 1 billion same target binding regions. In someembodiments, the different target binding regions can hybridize to atleast 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of thetranscripts of a transcriptome of an organism. In some embodiments, thedifferent target binding regions can hybridize to at least 0.01%, 0.02%,0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the transcripts of atranscriptome of an organism.

In one aspect the disclosure provides for a composition comprising: asolid support, wherein the solid support comprises a plurality ofoligonucleotides, wherein at least two of the plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein the cellular labels of the at least two of the plurality ofoligonucleotides are the same, and wherein the molecular labels of theat least two of the plurality of oligonucleotides are different.

In some embodiments, the plurality of oligonucleotide further comprisesa sample label. In some embodiments, the plurality of oligonucleotidesfurther comprises a target binding region. In some embodiments, thetarget binding region comprises a sequence is adapted to hybridize to atarget nucleic acid. In some embodiments, the target nucleic acidcomprises a plurality of target nucleic acids comprising at least 0.01%,0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of the transcripts of atranscriptome of an organism. In some embodiments, the target nucleicacid comprises sheared genomic DNA wherein the wherein the shearedgenomic DNA comprises at least 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, 90%, or 100% of the genes of a genome of an organism. In someembodiments, the target binding region comprises an oligo dT. In someembodiments, the at least two of the plurality of oligonucleotidescomprises a first oligonucleotide and a second oligonucleotide, whereinthe first oligonucleotide comprises a first cellular label and a firstmolecular label, wherein the first cellular label comprises a firstrandom sequence, a second random sequence, and a first linker labelsequence, wherein the first linker label sequence connects the firstrandom sequence and the second random sequence; and the first molecularlabel comprises a random sequence; and the second oligonucleotidecomprises a second cellular label and a second molecular label, whereinthe second cellular label comprises a third random sequence, a fourthrandom sequence, and a second linker label sequence, wherein the secondlinker label sequence connects the third random sequence and the fourthrandom sequence; and the second molecular label comprising a randomsequence, and wherein the first cellular label and the second cellularlabel are the same and the first molecular label and the secondmolecular label are different.

In one aspect the disclosure provides for a kit comprising anycomposition described herein and instructions for use.

In one aspect the disclosure provides for a method, comprising:contacting a sample with a solid support, wherein the solid supportcomprises a plurality of oligonucleotides, wherein at least two of theplurality of oligonucleotides comprises a cellular label and a molecularlabel, wherein the cellular labels of the at least two of the pluralityof oligonucleotides are the same, and wherein the molecular labels ofthe at least two of the plurality of oligonucleotides are different; andhybridizing the target nucleic acid from the sample to anoligonucleotide of the plurality of oligonucleotides.

In some embodiments, the sample comprises cells. In some embodiments,the sample is lysed prior to the hybridizing. In some embodiments, thehybridizing comprising hybridizing multiple copies of a same targetnucleic acid to the plurality of oligonucleotides. In some embodiments,the method further comprises reverse transcribing the target nucleicacid. In some embodiments, the method further comprises performing anoligonucletide amplification. In some embodiments, the amplifyingcomprises amplification using a method selected from the groupconsisting of: PCR, quantitative PCR, real-time PCR, and digital PCR, orany combination thereof.

In one aspect the disclosure provides for a A solid support comprising:a first oligonucleotide comprising: a first cellular label comprising afirst random sequence, a second random sequence, and a first linkerlabel sequence, wherein the first linker label sequence connects thefirst random sequence and the second random sequence; and a firstmolecular label comprising a random sequence; and a secondoligonucleotide comprising: a second cellular label comprising a thirdrandom sequence, a fourth random sequence, and a second linker labelsequence, wherein the second linker label sequence connects the thirdrandom sequence and the fourth random sequence; and a second molecularlabel comprising a random sequence, wherein the first cellular label andthe second cellular label are the same and the first molecular label andthe second molecular label are different. In some embodiments, the firstand second oligonucleotides further comprise identical sample indexregions. In some embodiments, the sample index region comprises a randomsequence. In some embodiments, the sample index region is 4-12nucleotides in length. In some embodiments, the cellular label isdirectly attached to the molecular label. In some embodiments, thecellular label and the molecular label are attached through a linkerlabel sequence. In some embodiments, the random sequence of the cellularlabel is from 4-12 nucleotides in length. In some embodiments, theconstant sequence of the cellular label is at least 4 nucleotides inlength. In some embodiments, the cellular label has a total length of atleast 12 nucleotides. In some embodiments, the cellular label furthercomprises one or more additional random sequences. In some embodiments,the cellular label further comprises one or more additional linker labelsequences. In some embodiments, the one or more additional linker labelsequences connect the one or more additional random sequences. In someembodiments, the random sequence of the molecular label is 4-12nucleotides in length.

In one aspect the disclosure provides for a composition comprising: asolid support, wherein the solid support comprises a plurality ofoligonucleotides, wherein at least two of the plurality ofoligonucleotides comprises: a cellular label, a molecular label; and atarget binding region; and a plurality of a target nucleic acids,wherein the cellular labels of the at least two of the plurality ofoligonucleotides are the same, and wherein the molecular labels of theat least two of the plurality of oligonucleotides are different.

In some embodiments, the target binding region comprises a sequence thatis adapted to hybridize to at least one of the plurality of targetnucleic acids. In some embodiments, the target binding region comprisesa sequence selected from the group consisting of: a random multimere.g., a random dimer, trimer, quatramer, pentamer, hexamer, septamer,octamer, nonamer, decamer, or higher multimer sequence of any length; agene-specific primer; and oligo dT; or any combination thereof. In someembodiments, the plurality of oligonucleotides comprises from 10,000 to1 billion oligonucleotides. In some embodiments, the plurality ofoligonucleotides comprises a number of oligonucleotides greater than thenumber of target nucleic acids of the plurality of target nucleic acids.In some embodiments, the plurality of target nucleic acids comprisesmultiple copies of a same target nucleic acid. In some embodiments, theplurality of target nucleic acids comprises multiple copies of differenttarget nucleic acids. In some embodiments, the plurality of targetnucleic acids are bound to the plurality of oligonucleotides. In someembodiments, the oligonucleotide further comprises a sample label. Insome embodiments, the plurality of oligonucleotides comprises auniversal label. In some embodiments, the universal label comprises abinding site for a sequencing primer. In some embodiments, the pluralityof oligonucleotides comprises a linker. In some embodiments, the linkercomprises a functional group. In some embodiments, the linker is located5′ to the oligonucleotide. In some embodiments, the functional groupcomprises an amino group. In some embodiments, the linker is selectedfrom the group consisting of: C6, biotin, streptavidin, primary amines,aldehydes, and ketones, or any combination thereof. In some embodiments,the solid support is comprised of polystyrene. In some embodiments, insolid support is magnetic. In some embodiments, the solid support isselected from the group consisting of: a PDMS solid support, a glasssolid support, a polypropylene solid support, an agarose solid support,a gelatin solid support, a magnetic solid support, and a pluronic solidsupport, or any combination thereofln some embodiments, the solidsupport comprises a bead. In some embodiments, the solid supportcomprises a diameter of about 20 microns. In some embodiments, the solidsupport comprises a diameter from about 5 microns to about 40 microns.In some embodiments, the solid support comprises a functional group. Insome embodiments, the functional group comprises a carboxy group. Insome embodiments, the functional group is selected from the groupconsisting of: C6, biotin, streptavidin, primary amines, aldehydes, andketones, or any combination thereof. In some embodiments, the cellularlabel comprises a plurality of cellular labels. In some embodiments, theplurality of cellular labels is interspersed with a plurality of linkerlabel sequences.

In one aspect the disclosure provides for a kit comprising: a firstsolid support, wherein the first solid support comprises a firstplurality of oligonucleotides, wherein the first plurality ofoligonucleotides comprises a same first cellular label, a second solidsupport, wherein the second solid support comprises a second pluralityof oligonucleotides, wherein the second plurality of oligonucleotidescomprises a same second cellular label, and instructions for use,wherein the first cellular label and the second cellular label aredifferent.

In some embodiments, oligonucleotides from the first plurality ofoligonucleotides and the second plurality of oligonucleotides comprisesa molecular label. In some embodiments, the molecular label of theoligonucleotides are different. In some embodiments, the molecular labelof the oligonucleotides are the same. In some embodiments, the molecularlabel of some of the oligonucleotides are different and some are thesame. In some embodiments, oligonucleotides from the first plurality ofoligonucleotides and the second plurality of oligonucleotides comprise atarget binding region. In some embodiments, the kit further comprises amicrowell array. In some embodiments, the kit further comprises abuffer. In some embodiments, the buffer is selected from the groupconsisting of: a reconstitution buffer, a dilution buffer, and astabilization buffer, or any combination thereof.

In one aspect the disclosure provides for a method for determining anamount of a target nucleic acid comprising: contacting a sample with asolid support, wherein the solid support comprises a plurality ofoligonucleotides, wherein at least two of the plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein the cellular labels of the at least two of the plurality ofoligonucleotides are the same, and wherein the molecular labels of theat least two of the plurality of oligonucleotides are different; andhybridizing the target nucleic acid from the sample to anoligonucleotide of the plurality of oligonucleotides.

In some embodiments, the sample comprises cells. In some embodiments,the sample is lysed prior to the hybridizing. In some embodiments, thehybridizing comprising hybridizing multiple copies of a same targetnucleic acid to the plurality of oligonucleotides. In some embodiments,the method further comprises amplifying the target nucleic acid. In someembodiments, the amplifying comprises reverse transcribing the targetnucleic acid. In some embodiments, the amplifying comprisesamplification using a method selected from the group consisting of: PCR,quantitative PCR, real-time PCR, and digital PCR, or any combinationthereof. In some embodiments, the amplifying is performed directly onthe solid support. In some embodiments, the amplifying is performed on atemplate transcribed from the solid support. In some embodiments, themethod further comprises sequencing the target nucleic acid. In someembodiments, the sequencing comprises sequencing the target nucleic acidand the molecular label. In some embodiments, the method furthercomprises determining an amount of the target nucleic acid. In someembodiments, the determining comprises quantifying levels of the targetnucleic acid. In some embodiments, the determining comprises countingthe number of sequenced molecular labels for the target nucleic acid. Insome embodiments, the contacting occurs in a microwell. In someembodiments, the microwell is made from a material selected from thegroup consisting of: hydrophilic plastic, plastic, elastomer, andhydrogel, or any combination thereof. In some embodiments, the microwellcomprises agarose. In some embodiments, the microwell is one microwellof a microwell array. In some embodiments, the microwell array comprisesat least 90 microwells. In some embodiments, the microwell arraycomprises at least 150,000 microwells. In some embodiments, themicrowell comprises at least one solid support per well. In someembodiments, the microwell comprises at most two solid supports perwell. In some embodiments, the microwell is of a size that accommodatesat most two of the solid support. In some embodiments, the microwell isof a size that accommodates at most one solid support. In someembodiments, the microwell is at least 25 microns deep. In someembodiments, the microwell is at least 25 microns in diameter.

In one aspect the disclosure provides for a method to reduceamplification bias of a target nucleic acid comprising: contacting asample to a solid support, wherein the solid support comprises aplurality of oligonucleotides, wherein at least two of the plurality ofoligonucleotides comprises a cellular label and a molecular label,wherein the cellular labels of the at least two of the plurality ofoligonucleotides are the same, and wherein the molecular labels of theat least two of the plurality of oligonucleotides are different; andhybridizing a target nucleic acid from the sample to the plurality ofoligonucleotides; amplifying the target nucleic acid or complimentthereof. sequencing the target nucleic acid or compliment thereof,wherein the sequencing sequences the target nucleic acid or complimentthereof and the molecular label of the oligonucleotide to which thetarget nucleic acid or compliment thereof is bound. determining anamount of the target nucleic acid.

In some embodiments, the hybridizing comprising hybridizing multiplecopies of a same target nucleic acid to the plurality ofoligonucleotides. In some embodiments, the determining comprisescounting a number of sequenced molecular labels for a same targetnucleic acid. In some embodiments, the counting counts the number ofcopies of the same target nucleic acid. In some embodiments, the samplecomprises cells. In some embodiments, the amplifying comprises reversetranscribing the target nucleic acid. In some embodiments, theamplifying comprises amplification using a method selected from thegroup consisting of: PCR, quantitative PCR, real-time PCR, and digitalPCR, or any combination thereof. In some embodiments, the amplifying isperformed directly on the solid support. In some embodiments, theamplifying is performed on a template transcribed from the solidsupport.

In one aspect the disclosure provides for a composition comprising amicrowell; a cell; and a solid support, wherein the solid supportcomprises a plurality of oligonucleotides, wherein at least two of theplurality of oligonucleotides comprises a cellular label and a molecularlabel, wherein the cellular labels of the at least two of the pluralityof oligonucleotides are the same, and wherein the molecular labels ofthe at least two of the plurality of oligonucleotides are different.

In some embodiments, the at least two of the plurality ofoligonucleotides further comprises a sample label. In some embodiments,the at least two of the plurality of oligonucleotides further comprisesa target binding region. In some embodiments, the target binding regioncomprises a sequence selected from the group consisting of: a randommultimer e.g., a random dimer, trimer, quatramer, pentamer, hexamer,septamer, octamer, nonamer, decamer, or higher multimer sequence of anylength; a gene-specific primer; and oligo dT; or any combinationthereof. In some embodiments, the plurality of oligonucleotidescomprises a universal label. In some embodiments, the universal labelcomprises a binding site for a sequencing prumer. In some embodiments,the solid support is comprised of polystyrene. In some embodiments, insolid support is magnetic. In some embodiments, the solid support isselected from the group consisting of: a PDMS solid support, a glasssolid support, a polypropylene solid support, an agarose solid support,a gelatin solid support, a magnetic solid support, and a pluronic solidsupport, or any combination thereofln some embodiments, the solidsupport comprises a bead. In some embodiments, the solid support has adiameter of about 20 microns. In some embodiments, the solid support hasa diameter from about 5 microns to about 40 microns. In someembodiments, the cellular label comprises a plurality of cellularlabels. In some embodiments, the plurality of cellular labels isinterspersed with a plurality of linker sequences. In some embodiments,the microwell is made from a material selected from the group consistingof: hydrophilic plastic, plastic, elastomer, and hydrogel, or anycombination thereof. In some embodiments, the microwell comprisesagarose. In some embodiments, the microwell is a microwell of amicrowell array. In some embodiments, the microwell comprises at leastone solid support per well. In some embodiments, the microwell comprisesat most two solid supports per well. In some embodiments, the microwellis of a size that accommodates at least one of the solid support and atleast one of the cell. In some embodiments, the microwell is of a sizethat accommodates at most one of the solid support and at least one ofthe cell. In some embodiments, the microwell is at least 25 micronsdeep. In some embodiments, the microwell is at least 25 microns indiameter. In some embodiments, the microwell is flat

In one aspect the disclosure provides for a device, comprising aplurality of microwells, wherein the plurality of microwells comprisesat least two microwells; and wherein each microwell of the plurality ofmicrowells has a volume ranging from about 1,000 μm³ to about 120,000μm³. In some embodiments, each microwell of the plurality of microwellshas a volume of about 20,000 μm³. In some embodiments, the plurality ofmicrowells comprises from about 1,000 to about 5,000,000 microwells. Insome embodiments, the plurality of microwells comprises about 100,000 toabout 200,000 microwells. In some embodiments, the microwells arecomprised in a single layer of a material. In some embodiments, at leastabout 10% of the microwells further comprise a cell. In someembodiments, at least about 10% of the microwells further comprise asolid support which comprises a plurality of oligonucleotides, whereinat least two of the plurality of oligonucleotides comprise a cellularlabel and a molecular label, wherein the cellular labels of the at leasttwo of the plurality of oligonucleotides are the same, and wherein themolecular labels of the at least two of the plurality ofoligonucleotides are different. In some embodiments, the solid supportsare magnetized.

In one aspect the disclosure provides for an apparatus comprising anydevice described herein, and a liquid handler.

In some embodiments, the liquid handler delivers liquid to the pluralityof microwells in about 1 second. In some embodiments, the apparatusdelivers liquid to the plurality of microwells from a single input port.In some embodiments, the apparatus further comprises a magnet. In someembodiments, the apparatus further comprises at least one of: an inletport, an outlet port, a pump, a valve, a vent, a reservoir, a samplecollection chamber, a temperature control apparatus, or any combinationthereof. In some embodiments, the apparatus comprises the samplecollection chamber, wherein the sample collection chamber is removablefrom the apparatus. In some embodiments, the apparatus further comprisesan optical imager. In some embodiments, the optical imager produces anoutput signal which is used to control the liquid handler. In someembodiments, the apparatus further comprises a thermal cycling mechanismconfigured to perform polymerase chain reaction (PCR) amplification ofoligonucleotides.

In one aspect the disclosure provides for a method of producing aclinical diagnostic test result, comprising producing the clinicaldiagnostic test result with any device or apparatus described herein. Insome embodiments, the clinical diagnostic test result is transmitted viaa communication medium.

In one aspect the disclosure provides for a device comprising: one ormore substrates further comprising one or more microwell arrays; whereinthe microwell arrays are used to perform multiplexed, single cellstochastic labeling and molecular indexing assays.

In some embodiments, the microwell arrays of the substrates comprisemicrowells arranged in a one dimensional or two dimensional arraypattern. In some embodiments, the two dimensional array pattern ofmicrowells is selected from the group including a square grid, arectangular grid, or a hexagonal grid.

In some embodiments, the microwells of the microwell arrays arefabricated using a well geometry selected from the group includingcylindrical, conical, hemispherical, rectangular, or polyhedral. In someembodiments, the microwells of the microwell arrays are fabricated usinga overall geometry that comprises two or more component geometriesselected from the group including cylindrical, conical, hemispherical,rectangular, or polyhedral. In some embodiments, the diameter of themicrowells in the microwell arrays is between about 5 microns and about50 microns. In some embodiments, the depth of the microwells in themicrowell arrays is between about 10 microns and about 60 microns. Insome embodiments, the center-to-center spacing between microwells in themicrowell arrays is between about 15 microns an about 75 microns. Insome embodiments, the total number of microwells in each of themicrowell arrays is between about 96 and about 5,000,000. In someembodiments, the one or more substrates are fabricated from a materialselected from the group including silicon, fused-silica, glass, apolymer, or a metal. In some embodiments, the one or more substrates arefabricated from agarose or a hydrogel. In some embodiments, themicrowell arrays further comprise surface features between microwellsthat surround the microwells or straddle the surface between microwells,and are selected from the group including domed, ridged, or peakedsurface features.

In one aspect the disclosure provides for a device comprising: asubstrate further comprising one or more microwell arrays; and amechanical fixture comprising a top plate, a bottom plate, and a gasket;wherein when assembled the substrate is positioned between the gasketand the bottom plate, the gasket forms a leak-proof seal with thesubstrate, and the top plate and gasket form one or more chambersencompassing the microwell arrays such that one or more cell samples andbead-based oligonucleotide labels may be dispensed into the chambers forthe purpose of performing multiplexed, single cell stochastic labelingand molecular indexing assays.

In some embodiments, the substrate comprises any one or more microwellarrays as described herein. In some embodiments, the gasket isfabricated from polydimethylsiloxane (PDMS) or a similar elastomericmaterial. In some embodiments, the top and bottom plates are fabricatedfrom aluminum, anodized aluminum, stainless steel, teflon,polymethylmethacrylate, polycarbonate, or a similar rigid polymermaterial.

In one aspect the disclosure provides for a device comprising: one ormore substrates further comprising one or more microwell arrays; and oneor more flow cells; wherein the one or more flow cells enclose or areattached to the one or more substrates, and include at least one inletport and at least one outlet port for the purpose of delivering fluidsto the microwell arrays; and wherein the device is used to performmultiplexed, single cell stochastic labeling and molecular indexingassays.

In some embodiments, the one or more substrates comprise any one or moremicrowell arrays as described herein. In some embodiments, each of theone or more flow cells further comprise a plurality of microarraychambers that interface with a plurality of microwell arrays such thatone or more samples may be processed in parallel. In some embodiments,the one or more flow cells further comprise a porous barrier or flowdiffuser to provide more uniform delivery of cells and beads to themicrowell arrays. In some embodiments, the one or more flow cellsfurther comprise dividers that divide chambers containing microwellarrays into subsections that collectively cover the same total arrayarea and provide for more uniform delivery of cells and beads to themicrowell arrays. In some embodiments, the width of fluid channelsincorporated into the device is between about 50 microns and 20 mm. Insome embodiments, the depth of fluid channels incorporated into thedevice is between about 50 microns and about 2 mm. In some embodiments,the one or more flow cells are fabricated from a material selected fromthe group consisting of silicon, fused-silica, glass,polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate (PMMA),polycarbonate (PC), polypropylene (PP), polyethylene (PE), high densitypolyethylene (HDPE), polyimide, cyclic olefin polymers (COP), cyclicolefin copolymers (COC), polyethylene terephthalate (PET), epoxy resin,metal, or a combination of these materials. In some embodiments, thedevice comprises a fixed component of an instrument system forperforming automated multiplexed, single cell stochastic labeling andmolecular indexing assays. In some embodiments, the device comprises aremovable component of an instrument system for performing automatedmultiplexed, single cell stochastic labeling and molecular indexingassays.

In one aspect the disclosure provides for a cartridge comprising: one ormore substrates further comprising one or more microwell arrays; one ormore flow cells or microwell array chambers; one or more sample orreagent reservoirs; and wherein the cartridge further comprises at leastone inlet port and at least one outlet port for the purpose ofdelivering fluids to the microwell arrays; and wherein the cartridge isused to perform multiplexed, single cell stochastic labeling andmolecular indexing assays.

In some embodiments, the one or more substrates comprise any one or moremicrowell arrays as described herein. In some embodiments, the one ormore flow cells or microwell array chambers interface with a pluralityof microwell arrays such that one or more samples may be processed inparallel. In some embodiments, the one or more flow cells or microwellarray chambers further comprise a porous barrier or flow diffuser toprovide more uniform delivery of cells and beads to the microwellarrays. In some embodiments, the one or more flow cells or microwellarray chambers further comprise dividers that divide the flow cells orchambers into subsections that collectively cover the same total arrayarea and provide for more uniform delivery of cells and beads to themicrowell arrays. In some embodiments, the width of fluid channelsincorporated into the cartridge is between about 50 microns and 200microns. In some embodiments, the width of the fluid channelsincorporated into the cartridge is between about 200 microns and 2 mm.In some embodiments, the width of the fluid channels incorporated intothe cartridge is between about 2 mm and 10 mm. In some embodiments, thewidth of the fluid channels incorporated into the cartridge is betweenabout 10 mm and 20 mm. In some embodiments, the depth of fluid channelsincorporated into the cartridge is between about 50 microns and about 10mm. In some embodiments, the depth of fluid channels incorporated intothe cartridge is between about 500 microns and 1 mm. In someembodiments, the depth of fluid channels incorporated into the cartridgeis between about 1 mm and about 2 mm. In some embodiments, the one ormore flow cells or microwell array chambers are fabricated from amaterial selected from the group consisting of silicon, fused-silica,glass, polydimethylsiloxane (PDMS; elastomer), polymethylmethacrylate(PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), highdensity polyethylene (HDPE), polyimide, cyclic olefin polymers (COP),cyclic olefin copolymers (COC), polyethylene terephthalate (PET), epoxyresin, metal, or a combination of these materials. In some embodiments,the device comprises a removable, consumable component of an instrumentsystem for performing automated multiplexed, single cell stochasticlabeling and molecular indexing assays. In some embodiments, thecartridge further comprises bypass channels or other design features forproviding self-metering of cell samples or bead suspensions dispensed orinjected into the cartridge. In some embodiments, the cartridge furthercomprises integrated miniature pumps for controlling fluid flow throughthe device. In some embodiments, the cartridge further comprisesintegrated miniature valves for compartmentalizing pre-loaded reagentsand for controlling fluid flow through the device. In some embodiments,the cartridge further comprises vents for providing an escape path fortrapped air. In some embodiments, the cartridge further comprisescomprise design elements for creating physical or chemical barriers thateffectively increase pathlength and prevent or minimize diffusion ofmolecules between microwells, wherein the design elements are selectedfrom the group consisting of: a pattern of serpentine channels fordelivery of cells and beads to the microwell array, a retractable platenor deformable membrane that is pressed into contact with the surface ofthe microwell array, or the release of an immiscible, hydrophobic fluidfrom a reservoir within the cartridge. In some embodiments, thecartridge further comprises integrated temperature control components oran integrated thermal interface for providing good thermal contact withan external instrument system. In some embodiments, the cartridgefurther comprises an optical interface or window for optical imaging ofthe one or more microwell arrays. In some embodiments, the cartridgefurther comprises one or more removable sample collection chambers thatare configured to interface with stand-alone PCR thermal cyclers and/orsequencing instruments. In some embodiments, the cartridge itself isconfigured to interface directly with stand-alone PCR thermal cyclersand/or sequencing instruments.

In one aspect the disclosure provides for an instrument systemcomprising: one or more flow cells or cartridges further comprising oneor more microwell arrays; and a flow controller; wherein the flowcontroller controls the delivery of cell samples, bead-basedoligonucleotide labeling reagents, and other assay reagents to themicrowell arrays, and the instrument system is used to performmultiplexed, single cell stochastic labeling and molecular indexingassays.

In some embodiments, the one or more microwell arrays are any describedherein. In some embodiments, the one or more flow cells are a fixedcomponent of the system. In some embodiments, the one or more flow cellsare a removable, consumable component of the system. In someembodiments, the one or more cartridges are removable, consumablecomponents of the system. In some embodiments, cell samples andbead-based oligonucleotide reagents are dispensed or injected directlyinto the cartridge by the user. In some embodiments, assay reagentsother than cell samples are preloaded in the cartridge. In someembodiments, the instrument system further comprises an imaging systemfor imaging the microwell arrays. In some embodiments, the instrumentsystem further comprises a cell or bead distribution system forfacilitating uniform distribution of cells and beads across themicrowell arrays, wherein the mechanism underlying the distributionsystem is selected from the group consisting of rocking, shaking,swirling, recirculating flow, low frequency agitation, or high frequencyagitation. In some embodiments, the instrument system further comprisesa cell lysis system wherein the system uses a high frequencypiezoelectric transducer for somicating the cells. In some embodiments,the instrument system further comprises a temperature controller formaintaining a user-specified temperature, or for ramping temperaturebetween two or more specified temperatures over two or more specifiedtime intervals. In some embodiments, the instrument system furthercomprises a magnetic field controller for use in eluting beads frommicrowells. In some embodiments, the instrument system further comprisesa computer or processor programmed to provide a user interface andcontrol of system functions. In some embodiments, the instrument systemfurther comprises program code for providing real-time image analysiscapability. In some embodiments, the real-time image analysis andinstrument control functions are coupled, so that cell and bead sampleloading steps can be prolonged or repeated until optimal cell/beaddistributions are achieved. In some embodiments, the instrument systemfurther comprises an integrated PCR thermal cycler for amplification ofoligonucleotide labels. In some embodiments, the instrument systemfurther comprises an integrated sequencer for sequencing ofoligonucleotide libraries, thereby providing sample-to-answercapability. In some embodiments, the cell samples comprise patientsamples and the results of the multiplexed, single cell stochasticlabeling and molecular indexing assay are used for clinical diagnosticapplications. In some embodiments, the cell samples comprise patientsamples and the results of the multiplexed, single cell stochasticlabeling and molecular indexing assay are used by a healthcare providerto make informed healthcare treatment decisions.

In one aspect the disclosure provides for software residing in acomputer readable medium programmed to perform one or more of thefollowing sequence data analysis: determining the number of reads pergene per cell, and the number of unique transcript molecules per geneper cell; principal component analysis or other statistical analysis topredict confidence intervals for determinations of the number oftranscript molecules per gene per cell; alignment of gene sequence datawith known reference sequences; decoding/demultiplexing of samplebarcodes, cell barcodes, and molecular barcodes; and automatedclustering of molecular labels to compensate for amplification orsequencing errors; wherein the sequence data is generated by performingmultiplexed, single cell stochastic labeling and molecular indexingassays.

What is claimed is:
 1. A composition comprising a particle, wherein the particle comprises a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.
 2. The composition of claim 1, wherein the particle is a bead.
 3. The composition of claim 2, wherein the bead is selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, antibody conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligodT conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.
 4. The composition of claim 1, wherein the plurality of oligonucleotides comprises DNA.
 5. The composition of claim 1, wherein at least 1,000 of the plurality of oligonucleotides comprise different molecular label sequences.
 6. The composition of claim 1, wherein at least 10,000 of the plurality of oligonucleotides comprise different molecular label sequences.
 7. The composition of claim 1, wherein the plurality of oligonucleotides comprises at least 10,000 oligonucleotides.
 8. The composition of claim 1, wherein each of the plurality of oligonucleotides further comprises at least one of: (a) a sample label; and (b) a universal label.
 9. The composition of claim 1, wherein the target-binding region comprises a sequence selected from the group consisting of a gene-specific sequence, an oligo-dT sequence, a random multimer, and any combination thereof.
 10. The composition of claim 1, wherein the molecular label sequences comprise random sequences.
 11. The composition of claim 1, wherein the particle support comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.
 12. The composition of claim 1, wherein (a) each of the plurality of oligonucleotides comprises a linker functional group, and (b) the particle comprises a solid support functional group; wherein the support functional group and the linker functional group are associated with each other.
 13. The composition of claim 12, wherein the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.
 14. The composition of claim 1, wherein the cellular label sequences of the plurality of oligonucleotides comprise at least 8 nucleotides.
 15. The composition of claim 1, wherein the molecular label sequences of the plurality of oligonucleotides comprise at least 8 nucleotides.
 16. A kit comprising a plurality of particles each comprising a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, and at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.
 17. The kit of claim 16, wherein the plurality of particles comprises a plurality of beads.
 18. The kit of claim 17, wherein the plurality of beads comprises at least 384 beads.
 19. The kit of claim 16, further comprising a microwell array, wherein each microwell of the microwell array comprises a particle of the plurality particles.
 20. The composition of claim 1, wherein the particle is a microparticle or a nanoparticle. 